RRM
2008 Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract:
Structured RNAs fold into complex globular molecules composed of helices and loops (internal, hairpin, or junction). The helices are comprised of Watson-Crick basepairs, while the loop regions tend to involve non-Watson-Crick basepairs. These loop regions may also participate in long-range RNA-RNA interactions that stabilize the 3D RNA structure. These long-range interactions generally involve non-Watson-Crick basepair interactions. Pairwise interactions between RNA bases can be classified geometrically by their base edges that interact (Watson-Crick, Hoogsteen, and Sugar Edge) and the relative orientation of their glycosidic bonds (cis or trans). Thus, there are 12 geometric basepairing families. A basetriple is a set of three nucleotides making two or three basepairs with one another, one of which may be a Watson-Crick pair. In a basetriple, at least one nucleotide must be involved in two basepair interactions. We used our “Find RNA 3D” (FR3D) search program to find all instances of basetriples. By searching symbolically for all instances of three bases in which a Watson-Crick or non-Watson-Crick basepair occurred between the first and second bse, and between the second and third base. We classified these according to the nature of the first and second basepairs. Combinatorially, 108 different kinds of basetriples were expected,12 cWW,12 tWW, 10 cWH, 10 tWH, 8 cWS, 8 tWS, 8 cHW, 8 tHW, 6 cHH, 6 tHH,4 cHS, 4 tHS, 4 cSW, 4 tSW, 2 cSH, and 2 tSH of which 64 were actually observed in the current database. From a reduced-redundancy set of atomic level x-ray crystal structures, the FR3D program found a large number of basetriple interactions, which were classified into geometric families based on the interacting edges. We determined the sequence signatures and distinct geometries within each geometric family. This information is useful in understanding and predicting allowed sequence variation between related organisms to better understand mutational data.
Keywords: RNA basepair, FR3D, geometric search program
Abstract not available online - please check the printed booklet.
Abstract:
MicroRNAs (miRNAs) are non-coding, endogenous; ~22nt long RNA sequences formed from primary transcripts in two steps by nuclear (Drosha) and cytoplasmic (Dicer) RNases. Many human miRNAs are expressed from introns of protein-coding transcripts, hence we wanted to explore if pre-mRNA splicing and miRNA processing are coupled. We constructed a minigene system with 4 exons and 3 introns from á-MHC gene that harbors miR-208 in intron 28. We induced mutations in the 5’ splice site and the branch site to study the effect of these mutations on pre-mRNA splicing. Additionally, we also incorporated mutations in the upper and lower helical regions of the stem-loop structure of pre-miRNA to study how these mutations affect miRNA processing. In in vivo system, mutations in the pre-miRNA structure had no affect on splicing. However, 5’ splice site mutation activated cryptic splicing. We are using in vitro assay systems to study intermediate splice products generated in both splicing and miRNA processing pathways using the above mentioned mutants.
Keywords: pre-mRNA splicing, Micro RNA processing
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
The clinical usefulness of aminoglycoside antibiotics has become limited due to an increase in antibiotic resistance. Resistance can occur if aminoglycosides become inactivated through enzymatic modification of their hydroxyl and amino groups, which can affect transport or ability of the compounds to bind to the target RNA. Recent approaches to overcome resistance have focused on simplifying the aminoglycosides and generating derivatives of the neamine core or 2-deoxystreptamine ring in order to maintain the minimal motif for binding, but avoid enzymatic modification. In this study, single-ring analogues of neamine were synthesized and ESI-MS was used to determine the binding affinity of the analogues to the decoding region A-site RNA from 16S rRNA (AS RNA). The data indicate that the 6-membered ring 2 moiety of neamine has weak binding to AS RNA, but there is no observed binding between the sugar moiety (ring 1) and AS RNA. In contrast, a synthetic ring 1 derivative shows moderate affinity to AS RNA (apparent Kd = 55 ± 4 µM) and the presence of a longer polyamine group at position N1 increases the binding affinity by 5.5 fold (apparent Kd = 10 ± 1µM). In contrast, blocking the polyamine group at the N1 position diminishes binding by five-fold. These data indicate that hydrogen-bond formation, as well as charge formation at the N1 position, is important for binding to AS RNA. The specificity of the tightest-binding derivative was tested using different RNA constructs (H69, H31, ssAS RNA, AS duplex RNA, and HS RNA). The preliminary data indicate that this neamine single-ring derivative has preferred binding to structured RNA as compared to ssRNA, as well as modest preference for AS RNA.
Keywords: aminoglycoside, antibiotics, ribosome
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Ribonuclease P (RNase P) is a Mg2+-dependent endoribonuclease which catalyzes the 5´ maturation of precursor tRNAs (ptRNAs). Although its primary function is conserved in all three domains of life, the subunit make-up of this ribonucleoprotein (RNP) varies. In bacteria, the holoenzyme is made up of one RNase P RNA (RPR) and one RNase P protein (RPP). In eukarya, RNase P contains an RPR and at least nine RPPs. Intermediate in complexity, archaeal RNase P comprises an RPR and four RPPs, which were computationally identified on the basis of homology to eukaryal counterparts. We employ archaeal RNase P as a model to address the roles of multiple RPPs in archaeal/eukaryal RNase P catalysis. By using recombinant Methanothermobacter thermautotrophicus (Mth) RPR and four RPPs (termed POP5, RPP21, RPP29, and RPP30), we have reconstituted the holoenzyme and performed single-turnover measurements. The maximal rate of ptRNA cleavage by the holoenzyme (i.e., RPR + 4RPPs) is ~300-fold faster (and at lower [Mg2+]) than that for the catalytic RPR alone. Using functional binary RPP complexes, we established that the RPR’s maximal kobs increases ~50-fold by addition of POP5-RPP30 but not at all by RPP21-RPP29, though the latter does enhance the kobs of RPR + POP5-RPP30 by 6-fold. Our data suggest that while POP5-RPP30 directly aids the phosphodiester bond-breaking step, the effects of RPP21-RPP29 manifest only when enabled by POP5-RPP30. Moreover, our findings that (i) a mutant Mth RPR with weakened binding of active-site Mg2+ is rescued upon addition of RPPs, and (ii) an N-terminal deletion mutant of RPP29 that fails to bind RPP21 (as judged by ITC and NMR experiments) is functional upon addition of RPR, illustrate the cooperative subunit interactions critical for driving RNase P towards its functional conformation. Together, these studies are beginning to shed light on the distinctive roles of RPPs in archaeal RNase P catalysis and the functional coordination among its subunits.
Keywords: Ribonuclease P, RNase P protein (RPP), single-turnover
Abstract:
RNase P is an essential endoribonuclease that removes the 5'leader sequence of precursor tRNAs (ptRNAs) during tRNA biogenesis. It functions as a ribonucleoprotein in all three domains of life. Previous studies have shown that in addition to ptRNAs, bacterial, plant, and human RNase P can also cleave model substrates built from two RNAs. For instance, an RNA molecule, called external guide sequence (EGS), could be designed to hybridize with a target RNA to form a bipartite ptRNA-like molecule that is recognized and cleaved by RNase P. Such an approach has been used to cleave and inactivate cellular target RNAs by artificially expressing the desired EGSs inside a cell. Since there is no reliable knock-down procedure in archaea, we are examining the feasibility of using archaeal RNase P for degrading cellular RNAs and thereby down-regulating their function. In vitro characterization of EGS-targeted RNA cleavage was performed using Pyrococcus furiosus (Pfu) RNase P reconstituted from the single RNA subunit, generated by in vitro transcription, and the four known protein subunits, which were co-overexpressed in Escherichia coli and co-purified as two binary complexes under native conditions. We determined that bipartite molecules, comprised of a target RNA substrate and EGS, are indeed efficiently cleaved by
Keywords: RNase P, External Guide Sequence, gene expression
Abstract:
Although it was previously thought that tRNAs are transcribed in the nucleus and unidirectly transported to cytoplasm for their function in protein synthesis. However, our lab showed that cytoplasmic tRNAs accumulate in the nucleus upon amino acid starvation in yeast and rat hepatoma cells (Shaheen and Hopper, 2005; Shaheen et al, 2007). Upon re-feeding, the accumulated cytoplasmic tRNAs are re-exported to the cytoplasm. Current data indicate that at least three members of β-importin family functionally participate in tRNA subcellular movement which includes three steps: 1) Los1, primary export of partially matured tRNA; 2) Mtr10, retrograde movement of tRNA into nucleus; 3) Msn5, re-export of fully mature tRNA to the cytoplasm. However, the biological consequences of the tRNA nuclear accumulation remain unclear. One possibility is that tRNA retrograde movement helps to down-regulate protein synthesis and change gene expression upon nutrient deprivation. To test this hypothesis, our strategy is to monitor changes in mRNAs associated with polysomes. The results of polysome profiles from msn5Δ mutants has no different relative to wild-type but mtr10Δ displays a translational elongation defect in responding nutrient stress. We have conducted microarray analyses of translating mRNAs from wild-type, msn5Δ, and mtr10Δcells under both fed and starved conditions. So far, I have learned that a subset of yeast mRNAs is affected.
Keywords: tRNA, transporter, microarray
Abstract:
In eukaryotes, 3?f end processing is an important post-transcriptional modification that results in mature mRNA. In plants, it is guided by three consensus elements, the far upstream element (FUE), near upstream element (NUE) and cleavage / polyadenylation site. This process is mediated by interactions between the protein and RNA as well as by protein-protein interactions. The key complexes involved in the poly (A) tail addition are Cleavage and Polyadenylation Specificity Factor (CPSF) and Cleavage and Stimulation Factor (CstF). These complexes are made up of multiple subunits. In mammals the cleavage stimulation factor is composed of three subunits, CstF50, CstF64 and CstF77. The present study focuses on the orthologues of mammalian CstF50 in Arabidopsis. CstF50 is a WD-repeat containing protein; Arabidopsis has two possible orthologues of CstF50. One of these orthologues (FY) has been shown to be involved in flowering while the other orthologue (At5g60940) has not been studied. In this regard, we show that AtCstF50 is essential and plays a different role from its mammalian counterpart. Further studies focus on its role in seed development.
In Arabidopsis FY is recruited to the mRNA by FCA, an RNA binding protein. FY in turn recruits 3?fend processing complex and mediates the polyadenylation reaction. We are testing this model using a tethering assay. Preliminary data in these lines will be presented.
References:
1. Li, Q. and A. G. Hunt (1997). The Polyadenylation of RNA in Plants. 115: 321-325.
2. Y Takagaki, J L Manley, C C MacDonald, J Wilusz, and T Shenk (1990) A multisubunit factor, CstF, is required for polyadenylation of mammalian pre-mRNAs.
3. Richard M. Amasino (2003). Flowering time: a pathway that begins at the 3?Ś end. vol 13 Issue 17.
Keywords: 3fend processing,CstF, FY, FCA, and tethering assay
Abstract:
Multiple peptide resistance factor (MprF) is a membrane bound protein which aminoacylates phophatidylglycerol using an elongator Lys-tRNALys. This lipid modification results in cellular permeability changes that confer resistance to cationic antibiotics and hence constitute a virulence factor in several pathogenic bacteria such as Listeria monocytogenes and Staphylococcus aureus. Previous studies have shown this method of membrane alteration plays an important role in the ability of L. monocytogenes to infect macrophages and epithelial cells. Many aspects of MprF involving pathogen fitness in infection have been studied however the importance of the protein in other physiological processes within the pathogen itself has been overlooked. Recently with the development of Phenotype MicroArrays by Biolog it is possible to perform a high-throughput screen for thousands of phenotypes conferred by a gene. Growth conditions testing phenotypes ranging from cell surface structure and transport functions to stress and repair functions are present in the wells of a microtiter plate that makes it effortless to determine significant effects of a gene on cellular processes. This study employs a Bacillus subtilis 1A100 with a marker-less deletion of mprF to compare to the wild type strain in a Biolog Phenotype MicroArray. Data obtained from this comparison sheds light on other roles of lipid remodeling in general cellular physiology in B. subtilis.
Keywords: MprF, lipid remodeling, tRNA
Abstract:
Class II prolyl-tRNA synthetase (ProRS) is a multidomain protein that attaches Pro to its cognate tRNAPro. In addition to an aminoacylation active site, most bacterial ProRSs also possess a separate editing domain (INS) that maintains fidelity of protein synthesis by hydrolyzing noncognate Ala mischarged onto tRNAPro. Escherichia coli (Ec) ProRS also misactivates Cys but is unable to hydrolyze Cys-tRNAPro via its INS editing domain. Instead, a small free-standing protein homologous to the INS domain (YbaK) has been shown to hydrolyze mischarged Cys-tRNAPro in trans, constituting a triple-sieve mechanism of editing. The gram-positive bacterium Enterococcus faecalis (Ef), which is an emerging pathogen in hospital-acquired infections, also encodes a YbaK homolog. Here, aminoacylation and hydrolytic editing activities of Ef ProRS and Ef YbaK are examined by performing cognate Pro charging, noncognate Cys mischarging, and Ala-, Ser- & Cys-tRNAPro deacylation assays. Ef ProRS possesses similar cognate Pro aminoacylation and noncognate Cys mischarging activity as Ec ProRS at 37 °C. Cys mischarging by Ef ProRS was abolished in the presence of Ef YbaK, consistent with the latter protein’s Cys-tRNAPro deacylase function. As expected, deacylation assays showed that Ef ProRS can hydrolyze mischarged Ala- but not Ser- or Cys-tRNAPro, while Ef YbaK can clear Cys-tRNAPro. In ongoing studies, the in vitro interaction between ProRS, YbaK and tRNAPro is being probed by fluorescence spectroscopy and the binding sites between them are being mapped by mass spectrometric protein footprinting. To gain further mechanistic insights into their biological roles, in vivo interactions of YbaK and other homologous proteins of unknown function, such as Ec YeaK, are being investigated using tandem affinity purification and mass spectrometry.
Keywords: ProRS, YbaK, Tandem affinity purification
Abstract not available online - please check the printed booklet.
Abstract:
Human tRNALys3 is used as the primer for HIV-1 reverse transcription. HIV-1 Gag and host cell lysyl-tRNA synthetase (LysRS) are both required for specific packaging of tRNALys into virions and these two proteins have been shown to interact in vitro. The capsid (CA) domain of Gag binds to LysRS with a similar affinity as full-length Gag. We have characterized the interaction between HIV-1 CA and human LysRS using truncation constructs, point mutants, and biophysical studies including mass spectrometry footprinting. The interaction involves helix 7 within the motif 1 dimerization domain of LysRS and the helix 4 region within the C-terminal domain of CA. Residues critical for maintaining this interaction in vitro and for packaging of LysRS in vivo have been identified. Synthetic peptides derived from the putative interaction helices bind to their respective protein partners and stabilized “stapled” peptides are currently being prepared and tested. Ongoing work is aimed at using these peptides as tools to identify molecules that disrupt this protein-protein interaction.
Keywords: LysRS packaging, LysRS-CA interactions
Abstract:
Pre-tRNA splicing is critical to the viability of eukaryotic cells. In vertebrates and yeast, the tRNA splicing endonuclease that cleaves the exon-intron junctions of pre-tRNA is composed of four proteins; Sen2p, Sen15p, Sen34p, and Sen54p. Splicing of pre-tRNA was demonstrated to occur in the nucleus of vertebrates such as Xenopus (Melton, et al., 1980) and humans (Paushkin et al., 2004). In contrast to vertebrate pre-tRNA splicing, yeast pre-tRNA splicing takes place in the cytoplasm on the surface of the mitochondria (Yoshihisa et al., 2003; 2007). The different location of pre-tRNA splicing machinery in yeast versus vertebrate cells raises the question of why the cell biology of this critical process differs between metazoans and yeast. To elucidate the spatial roles of the splicing endonuclease complex, we are directing the localization of the yeast pre-tRNA splicing machinery to the nucleus. To accomplish this, each protein of the splicing machinery has been fused in-frame to a nuclear localization signal and two tandem GFP molecules. To date each of the four components of the splicing endonuclease has been successfully relocated to the nucleus in cells that still contain the endogenous mitochondrial localized complex. Using heterokaryons we showed that none of the four subunits of the endonuclease shuttles out of the nucleus. Similar experiments with the tRNA ligase failed to deliver the majority of the protein into the nucleus. However, we have shown that the protein is not excluded from the nucleus and can still be used for the study. Our next goal is to determine whether the modified proteins maintain catalytic activity. Ultimately, we will determine the physiological consequences of yeast with defective cytosolic splicing machinery and a functional complex in the nucleus. We will accomplish this using either deletions and/or conditional mutations of the genes encoding the endogenous subunits of the splicing endonuclease complex.
Keywords: tRNA splicing
Abstract:
CPSF30 in Medicago.
Bobby Gaffney
CPSF30 is a component in 3’ RNA processing that has been well characterized in the model species Arabidopsis thaliana. In this study the gene for CPSF30 has been cloned out of Medicago truncatula and Medicago sativa. A large region within the gene exhibits significant conservation between A. thaliana and both medicago species. This area of the gene was cloned out and 5’ and 3’ RACE were used to get each full length gene. The Medicago genes were cloned into a protein expression vector. The vector was used to translate each gene into its corresponding protein and each protein was purified and dialyzed. CPSF30 in A. thaliana binds calmodulin and it was hypothesized that CPSF30 from both the Medicago species would also bind calmodulin based on the conservation of the amino acids between the model species and the Medicago species. This study shows that CPSF30 from both Medicago species does bind calmodulin. CPSF30 from A. thaliana also has been shown to possess three CCCH type zinc fingers, with the first implicated in RNA binding and the third in RNA cleavage. Amino acid alignments showed the zinc finger regions were conserved and it was hypothesized that CPSF30 from both Medicago species would bind RNA and cleave RNA. Electromobility shift assays were used to determine that RNA binding did occur. RNA nuclease activity in Medicago is still being tested. CPSF30 knockout studies in A. thaliana have yielded some consistent phenotypes that are still being characterized and explored. One such characteristic is an elevated resistance to oxidative stress. Currently CPSF30 knockouts are being produced in Medicago sativa that will enable further characterization of CPSF30 in an agriculturally relevant crop.
Keywords: CPSF30
Abstract not available online - please check the printed booklet.
Abstract:
The level of steady-state mRNA in the cell is a representation of the amount of mRNA synthesized by transcription minus the amount destroyed by the turnover pathway. In this study we provide evidence that mRNA decay rates can directly impact new mRNA synthesis. Eukaryotic mRNA turnover is initiated by the loss of the poly(A) tail, followed by decapping, and subsequent 5’-3’ exoribonuclease digestion. Mutations that block mRNA decapping result in dramatic stabilization of the mRNA in a transcriptional pulse-chase experiment. Shockingly, however, while the mRNA is five-fold more stable when decapping is impaired, there is no excess mRNA accumulation at steady state relative to WT. The incongruence between stabilization and steady-state levels is observed on multiple mRNAs, suggesting a widespread phenomenon. Moreover, while decapping mutants effect a step subsequent to deadenylation, the reduction in mRNA observed is on polyadenylated messages; implying an affect on newly generated transcripts. In addition, the impact on steady-state is specific to the decapping step and not seen when exonucleolytic digestion of the message is impaired. Our preliminary data suggests that this phenomena is a result of specific feedback between mRNA decapping and new mRNA transcription. This raises the possibility of a novel feedback loop, or retrocoupling event in which the proper destruction of the mRNA is required for the new generation of mRNA transcripts. Moreover, these findings demonstrate that the absolute levels of mRNA within the cell are kept at a finely controlled balance by both synthesis and destruction.
Keywords: mRNA decay, mRNA decapping, mRNA biogenesis
Abstract not available online - please check the printed booklet.
Abstract:
Human immunodeficiency virus type-1 (HIV-1) utilizes human cellular tRNALys in a critical step of its life cycle—the priming of reverse transcription of its single-stranded RNA into double-stranded DNA. For priming to occur, tRNALys must be annealed to the complementary 18-nucleotide site known as the Primer Binding Site (PBS) on the viral genome. Biological evidence suggests this process is performed by the viral Gag protein, the structural protein responsible for viral assembly. During the viral life cycle, Gag is proteolyzed into the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 proteins. A potent nucleic acid chaperone, NC plays a major role in restructuring RNA and DNA throughout the reverse transcription process. NC has been shown to anneal the tRNALys primer by promoting rearrangement of the PBS:tRNALys duplex to the thermodynamically most stable conformation. NC consists of two “zinc finger” motifs flanked by unstructured basic residues. Although both NC and Gag facilitate tRNA primer annealing to the PBS in vitro, we find reverse transcriptase extension is inhibited in the presence of Gag. Interestingly, in vivo studies have shown that Gag variants with zinc finger deletions or point mutations undergo premature reverse transcription, abolishing infectivity. In this work, similar Gag mutants were tested for their ability to bind and aggregate nucleic acids and to facilitate tRNA annealing and reverse transcription in vitro. We show that the nucleic acid chaperone activity of these Gag variants is robust and propose an explanation for Gag’s role in inhibiting premature reverse transcription.
Keywords: HIV-1, reverse transcription, RNA chaperone
Abstract:
It has been demonstrated that pRNA, a component of a switchable imitating DNA-packaging motor, can be used as a building block for bottom-up assembly in nanotechnology. The pRNA's structural versatility, coupled with its ability to form dimers, trimers, hexamers, and patterned superstructures via the interaction of two interlocking loops, makes it a promising tool for nanomachine fabrication, pathogen detection, and gene delivery. The presentation will focus on the approaches and methods for construction of multivalent therapeutic pRNA nanoparticles. The construction was based on the bottom-up assembly step by step with controllable structure and stoichiometry. The resulted polyvalent pRNA complex can deliver up to six kinds of therapeutics to specific cancer or viral infected cells as well as ailing cells involved in genetic diseases. Incubation of the RNA nanoparticles containing receptor-binding aptamer or folate resulted in cell binding and the transport of the chimeric pRNA/siRNA, pRNA/ribozyme, or drugs into cells subsequently causing gene silencing, modulating programmed cell death, or inhibiting viral replication. The efficiency was confirmed in animal trials. RNA 3-D design, circular permutation, folding energy alteration, and nucleotide modification were applied to generate stable RNA nanoparticles with low toxicity and to make the chimeric RNA complexes processed into siRNA by Dicer after delivery. Using such protein-free nanoparticles as therapeutic reagents would allow for long-term administration to avoid the induction of antibody due to repeated treatment of chronic diseases.
Keywords: phi29 DNA-packaging motor, therapeutic pRNA nanoparticles, bottom-up assembly
Abstract not available online - please check the printed booklet.
Abstract:
tRNAs are small RNA molecules that function in delivering amino acids to the ribosome during translation. We are studying how tRNA moves from its site of synthesis in the nucleus to its site of function in the cytoplasm. The yeast tRNA β-importin member, Los1, binds to end-processed tRNAs in the nucleus and exports them to the cytoplasm. However, since Los1 is unessential, additional nuclear export tRNA pathways must exist. Our lab identified another β-importin, Msn5, that functions in re-export of previously cytoplamsic tRNAs from the nucleus to the cytoplasm. Furthermore, we have shown that a temperature sensitive mutation of a third essential β-importin, Crm1, causes tRNA nuclear accumulation.
The question is: are Los1, Msn5, and Crm1 the only proteins involved in tRNA nuclear export? To study this, I am constructing yeast strains bearing multiple mutations of the known genes. Should the crm1-1 msn5Δ los1Δ triple mutant be viable, it will suggest there are other unidentified tRNA nuclear pathways. However, if the triple mutant is inviable, then possibly Los1, Msn5, and Crm1 are the only exportins involved. Previously, the los1Δ msn5Δ double deletion and the crm1-1 los1Δ double mutant were determined to be viable. I have constructed the crm1-1 msn5Δ::NAT double mutant and learned that it is also viable. However, interestingly, it displays increased temperature sensitivity compared with either single mutant. Therefore, the presence of these two mutations likely further impairs the export of tRNA from the nucleus. As this double mutant is viable, I am constructing the triple crm1-1 msn5 los1 mutant.
References:
Hopper, A.K. et al. (2008) A decade of surprises for tRNA nuclear-cytoplasmic dynamics. Trends Cell Biol. 18(3), 98-104.
Keywords: tRNA, export
Abstract:
Trm10 catalyzes SAM dependent N-1 methylation of G9 (m1G9) for at least 10 tRNA species in S. cerevisiae, including tRNAGly. The mechanism by which Trm10 methylates tRNA is unknown, and since there is no identifiable sequence homology between Trm10 and any other enzyme family, including other methyltransferases, the catalytic mechanism is likely novel. Several puzzling observations suggest that the Trm10 biological function is more complex than currently understood. Although m1G9 modification of tRNA is non-essential under standard biological conditions, recent results show that trm10Ä yeast strains are highly sensitive to 5-fluorouracil, a known anti-tumor agent for treatment of solid cancers; the reason for this observation is unknown. Moreover in yeast, TRM10 is a single copy gene, while multiple homologs of TRM10 are found in several higher eukaryotes, including at least three in humans.
Trm10 only modifies a subset of tRNA species in vivo. The sequence elements that define what tRNAs are substrates for Trm10 are unknown, and are not obvious from simple sequence comparison. We note, there are at least 12 other yeast tRNA species, including tRNAVal and tRNALeu, that have an unmodified G9. To investigate Trm10 substrate specificity, we have developed sensitive assays using three site-specifically labeled tRNA substrates. All of these substrates have a G residue at position 9, yet tRNAVal and tRNALeu are not substrates in vivo, while tRNAGly is a substrate for m1G9 modification. Trm10 activity with these substrates is being measured under steady-state conditions to determine parameters including kcat/KM, an indicator of substrate specificity. These results will be used as the foundation for identification of nucleotides necessary for tRNA specificity. We are also currently investigating the roles of several highly conserved amino acids in the Trm10 reaction by mutagenesis. Analysis of the resultant effects should provide insight into the molecular basis for Trm10 activity, a potentially novel methyltransferase enzymatic mechanism.
Keywords: tRNA, Trm10, yeast
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Riboswitches are gene regulatory elements found in the 5’ untranslated region of mRNA that operate without protein cofactors. Riboswitches can regulate the expression of corresponding genes by specifically recognizing and binding small molecules. Binding of the metabolite induces a conformational change that can either terminate transcription or prevent translation initiation. The ykkCD riboswitch is expected to control the expression of a multi-drug efflux pump that exports a variety of toxic drugs from the bacterial cell via transcriptional control. We will test whether transcription termination is the method of control by successively adding antibiotics to transcription reactions; antibiotics should trigger up-regulation of the efflux pump and gel electrophoresis will show if the pump is being transcribed. The rate of transcription can affect the regulatory function of the riboswitch if transcription is completed before metabolite binding induces the conformational change. We will examine how the rate of transcription compares to the rate of antibiotic binding and conformational change. We hope that this research may one day be useful in the fight against antibiotic resistance of infectious bacteria.
Keywords: riboswitch, gene regulation, transcription
Abstract:
tRNA processing is a multi-step process. Recent studies in the yeast S. cerevisiae have shown that pre-tRNA splicing occurs in the cytoplasm, and there is more than one tRNA nuclear export pathway. However, much remains to be learned about tRNA nuclear-cytoplasmic dynamics and pre-tRNA intron removal. We are employing candidate and genome-wide RNAomics approaches to identify novel gene products involved in pre-tRNA intron removal and pre-tRNA subcellular trafficking. So far, employing genome-wide RNAomics approaches, we learned that the growth phase of yeast cells appears to affect the fate of intron-containing tRNA. The findings suggest that there is cell cycle control over one or more of the tRNA nuclear export or for tRNA splicing. We are also attempting to design an in vivo biomarker for tRNA that will be used to follow the cellular movement of tRNA. This method employs a specific tRNA binding protein. If successful, this method could substitute for the laborious fluorescence in situ hybridization procedures currently used to track tRNA in cells and it will allow us to study tRNA subcellular dynamics in each of the >4000 yeast strains in the deletion collection.
References:
1. Hopper AK, Shaheen HH. A decade of surprises for tRNA nuclear-cytoplasmic dynamics. Trends Cell Biol. 18:98-104(2008).
2. Hopper, A.K. Cellular Dynamics of small RNAs. Critical Rev. Biochem. Mol. Biol. 41:3-19 (2006).
3. Weinert T, Hopper AK. tRNA traffic meets a cell-cycle checkpoint. Cell.; 131: 838-40(2007).
Keywords: tRNA processing
Abstract:
The yeast tRNAHis guanylyltransferase (Thg1) is the only known enzyme that catalyzes addition of nucleotides to a polynucleotide chain in the 3'-5' direction. In S. cerevisiae, Thg1 uses 3'-5' addition to add a single essential G residue (G-1) to the 5' end of tRNAHis. Since Thg1 is an essential enzyme that catalyzes a unique enzymatic activity, the Thg1 catalytic mechanism is of great interest. We have constructed a set of Thg1 variants in which highly conserved residues were altered individually to alanine, and analyzed the effects of these alterations on Thg1 activity, using both in vitro and in vivo assays (Jackman & Phizicky, 2008). We identified 12 residues that play significant roles in G-1 addition to tRNAHis in yeast, and a single residue that dramatically affects tRNA substrate selectivity of the enzyme. Transient kinetic approaches are being used to dissect the Thg1 reaction mechanism and to identify specific roles for each residue in catalysis.
Yeast Thg1 also catalyzes a second 3'-5' nucleotide addition reaction, templated 3'-5' polymerization of multiple nucleotides (Jackman & Phizicky, 2006). The universal requirement for G-1 for tRNAHis aminoacylation, and the occurrence of Thg1 homologs in all sequenced eukaryotes, suggests that G-1 addition to tRNAHis is a conserved role of Thg1 in eukaryotes. In contrast, the physiological function of 3'-5' polymerization is not known, in yeast or in any organism. However, Thg1-related cell-cycle defects are observed in both yeast and human cells that are not obviously related to a role for the enzyme in processing tRNAHis, and moreover, Thg1 homologs have been identified in several archaealand bacteria that do not need enzymatic G-1 addition to tRNAHis. To determine whether there are alternative roles for Thg1, possibly related to its ability to catalyze 3'-5' polymerization, we have compared the 3'-5' nucleotide addition activities of Thg1 and several of its homologs. We have observed characteristic differences in catalytic activity of Thg1 from different domains of life that can be exploited to further probe the role of Thg1 in yeast and other organisms.
References:
Jackman JE, Phizicky EM. 2006. tRNAHis guanylyltransferase catalyzes a 3'-5' polymerization reaction that is distinct from G-1 addition. Proc Natl Acad Sci U S A 103:8640-8645.
Jackman JE, Phizicky EM. 2008. Identification of critical residues for G-1 addition and substrate recognition by tRNA(His) guanylyltransferase. Biochemistry 47:4817-4825.
Keywords: tRNA, Thg1, enzyme mechanism
Abstract not available online - please check the printed booklet.
Abstract:
Total internal reflection fluorescence (TIRF) microscopy is used to image polycatalytic supramolecular complexes, dubbed “spiders,” moving across defined substrate tracks on tiles of DNA origami, long single-stranded DNA molecules folded into a 2-dimensional landscapes by hundreds of short DNA “staples.” Each spider is composed of a streptavidin body and three 8-17 deoxyribozyme-bearing legs which allow for motion across the single-stranded DNA substrate in a self-repelling random walk via the catalytic activity of its legs upon a site following a single RNA base in the substrate sequence. By registering the images of diffraction-limited point spread functions (PSFs) of emission from Cy3 and Cy5 labels on the spider and origami, respectively, and fitting Gaussian functions to these PSFs, spider positions on the origami track are measured with low-nanometer spatial precision to monitor the spider’s motion over time. Using this technique, individual spiders are observed to traverse 100-nm linear tracks in the presence of zinc ions, which are essential for 8-17 deoxyribozyme activity, but do not exhibit any net displacement in the absence of zinc ions. The spiders also traverse more complicated substrate tracks containing approximately 90-degree turns. These experiments are initial steps towards nanoscale control of dynamic artificial molecular assemblies. Preliminary results and future directions are discussed.
Keywords: Total internal reflection fluorescence, Single particle tracking, DNA origami
Abstract:
Enzymes processing nucleic acids usually are studied by discontinuous gel-electrophoresis assays using radioactive or fluorescence labeled substrate molecules. Besides the fact that the labels can perturb the reaction and pose a health risk to the investigators, the assays usually involve extra experimental steps: quenching the reaction, separation, visualization and quantification of the products. Here we describe real-time and simple optical assays to monitor nucleic acid strand-exchange, DNA/RNA cleavage and elongation of DNA. The methods take advantage of the property of some guanine-reach oligonucleotides to adopt monomolecular quadruplex conformation in the presence of certain cations. The conformation is characterized by significant absorption in long-wavelength range of the ultraviolet region where usually other secondary structures are transparent. The “signal” oligonucleotide is incorporated into reactant duplex which is released into solution upon an enzyme activity. The release is accompanied by fast quadruplex formation and ultraviolet absorption in long-wavelength range.
Keywords: DNA strand-exchange, DNA cleavage, DNA Quadruplex
Abstract:
Splicing is an essential and highly complex process in eukaryotic gene expression. It is catalyzed by the spliceosome, which is a dynamic assembly of five small nuclear RNAs (snRNAs) and a large number of proteins. Out of five snRNAs, the highly conserved U2 and U6 snRNAs are required in both steps of splicing. The U2-U6 snRNA complex forms the active site of the spliceosome and has been shown to undergo splicing-related catalysis in the absence of proteins. Our single-molecule data of yeast U2-U6 snRNAs has shown a Mg2+-induced conformational change consisting of a two-step process, which may be involved in spliceosomal activation in vivo. In contrast to yeast, human U2 and U6 snRNAs contain a large number of post-transcriptional modifications and CA–GU wobble base pairs as opposed to the UU wobble base pairs found in yeast. According to recent studies, these differences make human snRNAs more stable than that of yeast lending to the possibility of different activation mechanisms between the two species.
In order to understand and compare the catalytic mechanisms, we use single molecule florescence to characterize the conformational changes of the human U2-U6 complex in the presence and absence of modifications using Mg2+ as a divalent metal ion. Our FRET data clearly shows a Mg2+-induced conformational change of the unmodified human U2-U6 complex as we previously observed in yeast. We are currently studying the effects of post-transcriptional modifications of the human snRNAs on these dynamics.
Keywords: Single-molecule, Spliceosome, U2-U6 snRNAs
Abstract:
Nucleoside modifications are a universal feature of transfer RNA, but their levels are influenced by various factors such as light exposure, dark, temperature, water stress, etc. In this report, we present data that suggests differential modification levels in total tRNA isolated from Cucumis sativus cotyledons grown under either 12 hr dark and 12 hr light conditions, total dark or dark grown but exposed to light 24 hr before harvesting. Comparative nucleoside analysis of total tRNA isolated from these plant samples revealed interesting features. While all the plant samples exhibited nucleoside modifications such as pseudouridine, 2’-O-methyluridine, 1-methylguanosine or 2’-O-methylguanosine, and 2’-O-ribosyladenosine, completely dark grown plants exhibited elevated levels of 1-methyladenosine, N4-acetylcytidine or N2–methylguanosine. On the other hand, plants grown under 12 hr dark and 12 hr light conditions as well as the dark grown but exposed to 24 hr light exhibited higher levels of 2’-O-methylcytidine. The dark grown exposed to light also exhibited higher levels of N4-acetylcytidine or N2–methylguanosine. Several peaks were unidentifiable in the respective chromatograms in all the plant samples. Further analysis involves collecting fractions and subsequent analysis by mass spectrometry to determine the identity of these modifications. The implications associated with increased levels of nucleoside modifications in response to extreme environmental conditions will be discussed.
References:
Jayabaskaran C. and Hande S. Plant Growth Regulation 1995, 16: 73-81.
Pomerantz S.C. and McCloskey J. 1993, 193, 796-824.
Keywords: Nucleoside modifications, tRNA, Cucumis sativus
Abstract:
A growing list of naturally leaderless mRNAs has been reported to occur in all three domains of life. These leaderless mRNAs lack a 5' untranslated region (UTR) and are unable to establish the SD-ASD base-pairing between mRNA and 16S rRNA. Nevertheless, some of these leaderless mRNAs are efficiently translated. In addition, some mRNAs have very short untranslated regions (sUTR, length = 1-4 nt) upstream to the start codon. Leaderless cI-lacZ reporter constructs with added sUTRs were used to study the effect of sUTRs on expression. We observed a decrease in expression upon addition of sUTRs, with less than 5% expression when the sUTR length was 6nts. Further investigation revealed a direct correlation between decreased expression and decreased ribosome binding to the 5’-recessed AUG start codon. Additional investigation focused on the influence of the nucleotide composition of a ‘-1 triplet’ added immediately upstream of the start codon. The expression data shows that the influence of the ‘-1 triplet’ nucleotide composition varies between minimal to significant influence. Filter binding assays suggest that the influence of the ‘-1 triplet’ sequence on initial mRNA-ribosome association was minimal. Toeprint assays, that measure stable ternary complex, show a direct correlation between decreased expression and decreased ribosome binding to the 5’-recessed AUG start codon. Competition toeprint assays reveal less stability for the ‘-1 triplet’ mRNAs with low rates of ternary complex formation. Photo-crosslinking assays were used to further investigate the ribosome-5’-AUG interactions contributing to the formation of stable ternary complex. Our results suggest that E. coli ribosomes interact with a 5’-AUG at several sites and ultimately form stable ternary complexes with the 5’-AUG at ribosomal P-site.
Keywords: Leaderless mRNA, Translation initiation, mRNA ribosome interactions
Abstract:
RNA interference (RNAi) is a mechanism that enables sequence specific regulation of gene expression at the RNA level. Exogenous long double-stranded RNAs function as initiating substrates for the RNAi pathway. They are cleaved into short interfering RNA (siRNA) duplexes by an endogenous RNase III-type enzyme called Dicer. The siRNA duplexes get loaded into the effector RNA Induced Silencing Complex (RISC) by Dicer and are unwound in a strand specific manner during RISC assembly. This single-stranded siRNA guides RISC to effect sequence specific degradation of target mRNA 1.
RISC assembly has been extensively studied in cell extracts of embryos of the fruit fly Drosophila 3, but little is known about the interaction of siRNAs, Dicer, and RISC proteins in mammalian cells. To identify the component proteins of human RISC and other siRNA containing complexes, we performed native (non-denaturing polyacrylamide) gel-shift assays after incubating cytosolic HeLa cell extract with radiolabeled siRNA duplexes for varying time periods under buffered conditions. Three distinct siRNA-protein complexes, R1, R2, and R3, with distinct gel mobility were observed at all incubation times, but with varying relative abundances. On studying the kinetics of these complexes, the following rates were obtained: rate of formation of R1 and R3 =7.83 ± 4.50 e-4 sec -1 and 4.33 ± 0.83 e-4 sec -1 respectively, and the rate of disappearance of R2 =2.23 ± 1.00 e-3 sec -1. These complexes were subjected to mass spectrometry analysis. In addition, Western Blotting was performed on these complexes for certain proteins known to be involved in the RNAi pathway. Dicer was found to be present in R1, and the content of Dicer in the complex decreased with increasing incubation time. This is consistent with the hypothesis that dicer gets displaced from the mature RISC complex 1, 2. The other components of RISC are yet to be identified.
References:
1. Witold Filipowicz (2005) RNAi: The Nuts and Bolts of the RISC Machine, Cell, Vol. 122, 17–20.
2. ,Hutvágner G, Zamore PD (2002) RNAi: nature abhors a double-strand, Curr Opin Genet Dev. 12(2):225-32.
3. John W. Pham, Janice L. Pellino, Young Sik Lee , Richard W. Carthew, and Erik J. Sontheimer (2004), A Dicer-2-Dependent 80S Complex Cleaves Targeted mRNAs during RNAi in Drosophila, Cell, Vol. 117, 83–94
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Keywords: RNAi, siRNA, RISC
Abstract:
Translation initiation in bacteria involves recruitment and binding of mRNA to ribosomes through a complementary Shine-Dalgarno (SD) – anti Shine-Dalgarno (ASD) interaction. We have added saturating amounts of a DNA oligonucleotide, complementary to the ribosome’s ASD sequence, to ribosomes, thereby eliminating its availability to the mRNA SD sequence. Despite having neutralized what is generally thought to be the primary mechanism for ribosome binding, the ribosomes still bound to the test mRNA. Data from primer extension inhibition (toeprint) assays suggests that blocking the SD-ASD interaction does not eliminate mRNA-ribosome binding and suggests that additional signals in the mRNA exist to promote ribosome binding. Additional evidence suggests that secondary signals in the mRNA not only promote ribosome binding but also allow translation. This was accomplished by mutating the SD sequence in the untranslated lac leader to its complementary nucleotide sequence, thereby eliminating its complementarity to the anti-SD found on the 30S ribosomal subunit. The lac leader region, both with and without SD mutations, was fused to a lacZ reporter gene and was assayed for the production of beta-galactosidase. Growth on plates containing X-gal indicated that presence of lacZ mRNA with the mutated SD still promoted translation initiation in E. coli. To further investigate the presence of additional signals, a series of deletions to the 5’UTR, both with and without the SD mutations, were fused to a lacZ reporter and transformed into E. coli. The constructs will be assayed for beta-galactosidase activity and for in vitro ribosome binding. The goal of this project is to identify the nucleotide sequences responsible for ribosome binding and expression in the absence of a SD-ASD interaction.
Keywords: ribosome binding, translation initiation, mRNA untranslated leader
Abstract:
Ribosomes are responsible for catalyzing protein synthesis in all cells. Assembly of the four rRNAs and 78 ribosomal proteins in eukaryotic ribosomes requires a large molecular machinery of ~200 proteins and dozens of RNAs. GTPases are one class of assembly factors that is conserved from bacteria to humans and of specific interest due to its potential regulatory role. Previous work has demonstrated that the essential GTPase Bms1 promotes assembly of a pre-40S ribosomal particle essential for rRNA processing (1). Here, we focus on the mechanism by which the GTPase activity within Bms1 is stimulated by an unusual intramolecular GTPase-activating (GAP) domain. We have devised an assay in which GTPase and GAP domains are provided on separate molecules to identify the GAP domain. This assay has identified a 150 amino acid region that provides GTPase stimulation in trans. Interestingly, this region includes at least two essential amino acids as well as a conserved phosphorylation site, which regulates the activity of Bms1 in vivo. We are currently testing the role of the essential amino acids for GTPase activation as well as other functions of Bms1. Additionally, our experiments indicate the GAP domain is more effective when provided in trans than in cis, indicating the alignment of the domains in the free Bms1 protein is not optimized. The signal that brings the two domains into contact will be probed to better understand and potentially block the contact leading to activation. Investigating the interaction of the domains will lead us to determine whether binding to pre-ribosomes influences the interaction between the GAP and GTPase domains of Bms1.
References:
1. Karbstein, K., Jonas, S., Doudna, J. (2005) An Essential GTPase Promotes Assembly of Preribosomal RNA Processing Complexes. Mol. Cell 20, 633-643.
Keywords: Small subunit assembly, GTPase, ribosome biogenesis
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Thermodynamic parameters are reported for duplex formation of thirty RNA duplexes containing group II single nucleotide bulge loops were optically melted in 1M NaCl, and the thermodynamic parameters H°, S°, G°37, and TM for each sequence were determined. Group II bulge loops have two or more consecutive identical nucleotides at the bulge position such that there is ambiguity as to which nucleotide is the bulge. Sequences from this study were combined with sequences from a previous study (Znosko et al., (2002) Biochemistry 41, 10406-10417), thus examining all possible group II single nucleotide bulge loop sequences. The combined data were used to develop a nearest-neighbor model to predict the free energy of an RNA duplex containing a single nucleotide bulge. The free energy increments for the introduction of a group II bulge loop range from 0.9 and 7.3 kcal/mol. The introduction of the bulge was found in all cases to destabilize the duplex. As observed previously for Group I bulge loops (Blose et al., (2007) Biochemistry 46, 15123-15135), neither the identity of the bulge nor its nearest neighbor had an effect on the destabilization of the duplex by the bulge. The influence of the bulge on the duplex stability was primarily affect by non-nearest-neighbor interactions. Specifically, there is a direct correlation between the destabilization of the duplex and the stability of the stems adjacent to the bulge.
Keywords: bulge loop, stability, thermodynamics
Abstract:
Caged 5´-bridging phosphorothiolates to analyze ribozyme cleavage
Joy Krishna Maity, Tao Han, Ahmed Al-Harbi & Subha R Das*
Department of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213-3890, USA
Synthetic analogues of oligonucleotides are essential tools for probing mechanisms of enzymes that act on nucleic acids. Among the various modifications to nucleic acids, replacement of the phosphoryl oxygen atoms with sulfur has proven to be particularly useful in elucidating ribozyme mechanisms. Of the four possible phosphoryl oxygen atoms, replacement of the 5'-oxgen remains a particularly challenging modification. Access to RNA containing a 5'-S-phosphorothiolate (5'-PS) linkage requires synthesis of modified monomers and following synthesis, the 5'-PS linkage is 105 more labile than the native RNA phosphodiester. In order to preserve the integrity of the RNA containing the 5'-PS linkage the vicinal 2'-hydroxyl requires protection that persists through solid-phase synthesis and deprotection of the RNA. The photo-labile or 'caging' o-nitrobenzyl (oNBn) group fulfills the role of such an orthogonal protection in RNA synthesis. Here we describe the synthesis of three different classes of nucleoside phosphoramidites (i) containing a 5'-sulfur atom, (ii) containing a oNBn-protected 2'-hydroxyl group and (iii) containing both 5'-sulfur atom as well as (oNBn)-protected 2'-hydroxyl group. Combinations of these phosphoramidities can be used to generate 5'-S-phosphorothiolate containing RNA that are useful as hyperactivated substrates to analyze ribozyme mechanisms.
Keywords: Oligonucleotide, Phosphorothiolate, Synthesis; Ribozyme
Abstract:
All tRNAHis contain an additional G residue (G-1) that is necessary for aminoacylation by the cognate synthetase (HisRS). G-1 is added post-transcriptionally in eukaryotes, across from a universally conserved A at position 73, by an essential enzyme, the tRNAHis guanylyltransferase (Thg1).
The known physiological substrate for Thg1, tRNAHis, shares the highly conserved structure of all tRNAs, yet must be individually recognized by Thg1 for selective G-1 addition; the molecular basis of this recognition is not entirely understood. Insight into tRNA recognition by HisRS has been achieved using small RNA stem-loop substrates. Consequently, we measured steady-state kinetic parameters for Thg1 activity with two small RNA stem-loops. Following nomenclature used for HisRS, the first substrate (micro-His) mimics the 7 bp acceptor stem of tRNAHis, and the second substrate (mini-His) mimics the acceptor stem and 5 bp T stem, which coaxially stack in full-length (FL) tRNA. Thg1 exhibits nearly identical kcat values for G-1 addition with micro-His and mini-His. The measured Km’s for these substrates reveal an intriguing trend since Km for mini-His is ~5-fold greater than Km for FL tRNAHis, yet Km does not continue to increase as the substrate is shortened further. Instead Km for micro-His is surprisingly low, similar to that measured for FL tRNA.
Thg1 catalyzes a second biochemical reaction, 3’-5’ addition of multiple nucleotides to tRNA substrates where A73 has been substituted with C. Therefore, we made analogous substitutions in the small RNAs and found that for both substrates, substitution of C resulted in ~30-fold increase in kcat/Km compared to its A-containing counterpart, mainly driven by an increased kcat. Since these substrates are otherwise identical, these data suggest that increased catalytic activity is attributable to an enhanced rate under conditions where Watson-Crick base pairs are formed. Since kcat/Km for C-containing micro-His is within 2-fold of that observed for wild-type FL tRNAHis, non-tRNA species could be catalytically competent enough to act as physiologically relevant Thg1 substrates in vivo.
Keywords: Thg1, tRNAHis, catalysis
Abstract not available online - please check the printed booklet.
Abstract:
RNA oligomers containing two single bulge loops were analyzed to determine the thermodynamic parameters (ΔH o, ΔS o, ΔG 37 oand T M) through optical melting in 1 M NaCl. The second bulge loop was evaluated in 16 duplexes where the bulge was positioned on the same side of the duplex as well as on the complimentary strand. The duplex between the bulge loops, the inner duplex, was varied in length and sequence in addition to the orientation of the bulge. Inner duplexes consisting of two to four A-U and G-C base were evaluated with both bulges placed on the same side. Duplexes with the bulges on opposite strands that were composed of two to six G-C or A-U base pairs or a combination of G-C and A-U pairings were also examined. The thermodynamic data was compared to the current bulge loop prediction model (Blose, Silverman, and Bevilacqua, Biochemistry 2007, 4232-4240). The Blose model is not accurate in determining the destabilizing effect of two single base bulges because the second bulge affects the duplex differently from the first bulge. However, the Blose model does accurately predict that the bulge becomes less destabilizing as the adjacent stem becomes less stable. A larger data set should be examined in order to determine the effect of a second single base bulge loop in a duplex.
References:
Blose J.M., Silverman S.K., Bevilacqua P.C. (2007) A Simple Molecular Model for Thermophilic Adaption of Functional Nucleic Acids. Biochemistry 2007, 4232-4240.
Keywords: RNA thermodynamics, bulge loops
Abstract:
The T box family of genes are primarily found in Gram-positive bacteria and are characterized by highly conserved primary and secondary structural elements in the 5’ untranslated region. The expression of the genes (typically related to amino acid biosynthesis) is up regulated by the cognate tRNA interaction with the conserved 5’ untranslated region of the nascent mRNA in at least two positions. One of the interactions involves base pairing of the acceptor end of the tRNA with four base pairs of the bulge in a conserved secondary structural element known as the antiterminator. The antiterminator consists of two helices, A1 and A2 separated by a seven-nucleotide bulge and a closing loop in the A2 helix. The tRNA affinity of antiterminator model RNA with different loops was investigated. The effects of divalent metal ions and loop stability on structure and affinity of the antiterminator model RNA were investigated using computational, spectroscopic and molecular biology methods. The results indicate that divalent metal ions are required for maximal tRNA affinity and that this effect is modulated by the stability of the loop indicating that the overall stability (and possibly flexibility) of the antiterminator plays a role in tRNA binding.
Keywords: T box genes, RNA stability, structure-function, binding
Abstract:
New paradigms for the roles of RNA are emerging and it is clear that RNA plays a much larger role than previously identified. These discoveries are fueled through greater understandings of RNA structure and function relationships. We aim to understand better the contributions of specific structural features, such as the 2'-hydroxl group, to RNA function through atomic substitutions to RNA. We aim to test the hypothesis that RNA containing a 2'-deoxy-2'-chloro (2'-Cl) analogue is unable to be cleaved by self-cleaving ribozymes, yet still retains the inherent conformational flexibility of the natural ribo-sugar phosphate backbone of RNA. Starting with uridine (U), we synthesized in four steps the protected form of 2'-Cl-U required for solid phase RNA synthesis. The stem and loop region of the U1-RNA sequence will then be synthesized for co-crystallization with the U1A protein. The U1A protein was obtained by fusion to a (His)6-GST tag containing a TEV cleavage site for purification. It was expressed in E.coli and purified by Ni+2 chromatography, followed by cleavage of the fusion protein using TEV. We synthesized U1-RNA containing 2'-Cl-U, and aim to co-crystallize it with the U1A protein, and solve the structure for comparison with the known structures of the natural co-complex. This will determine how well the 2'-Cl substituents mimic the natural ribo 2'-OH residues in U1-RNA. This may also help to determine what structural features of the RNA backbone conformation are important for RNA-protein recognition. Further studies will be done where the 2'-Cl-U will be incorporated into self-cleaving ribozymes to prevent cleavage. Crystallization of these non-cleaved ribozymes would give further insight to the mechanism of their cleavage.
Keywords: ribozyme, RNA analogue
Abstract:
Beta-thalassemia is an inherited disorder of hemoglobin production that results from the presence of a premature termination codon in the body of beta-globin mRNA. In murine erythroleukemia (MEL) cells, PTC-containing beta-globin mRNA is degraded by endonuclease cleavage and results in the accumulation of metastable mRNA decay intermediates. An earlier report suggested that these intermediates possess a cap-like modification at 5?f end [Lim, S.K., and Maquat, L.E. 1992. EMBO J. 11:3271-3278]. The first part of this study examined the cap status of these decay intermediates by their recovery with an immobilized monoclonal antibody to the trimethylcap or a cytoplasmic cap binding protein eIF4E. Both full-length mRNA and the smaller decay products were recovered, supporting the presence of a true cap. This was confirmed by cap analog competition for binding to eIF4E and susceptibility to hydrolysis by the cellular decapping enzyme Dcp2. Although mammalian capping enzyme (CE) is generally thought to be restricted to the nucleus, a population of cytoplasmic CE was identified by Western blotting, &alpha-32P GMP labeling of the active site intermediate and fluorescence microscopy. Cytoplasmic CE sediments at 140-200 kDa on glycerol gradients and immunoprecipitated cytoplasmic CE complex converts a 5?f-monophosphate (but not a 5?f-hydroxyl) RNA to 5?f GpppX. The recovery from stress was used to evaluate the biological function of cytoplasmic capping enzyme. Arsenite causes a generalized inhibition of translation and the ability of cells to recover from arsenite stress was reduced by expression of inactive capping enzyme in a form that is restricted to the cytoplasm. These data support a role for capping in the cytoplasm and suggest that some mRNAs may be stored in an uncapped state.
Keywords: endonuclease-mediated mRNA decay, cytoplasmic capping, cell stress
Abstract:
The glmS riboswitch occurs in all gram-positive bacteria exerting feedback control over production of glucosamine-6-phosphate (GlcN6P) that is used in cell-wall synthesis. Recent biochemical and structural analyses suggest that GlcN6P binds to the RNA and act s directly to catalyze RNA cleavage. Through analyses of the cleavage reaction with normal and hyperactivated 5'-S-phosphorothiolate substrates as well as differing cofactors, we are investigating the mechanistic role of the putative GlcN6P cofactor. In addition, to determine the effectiveness of a GlcN6P cofactor in catalysis of RNA cleavage we are using in vitro selection (SELEX) methods to identify GlcN6P-dependent DNAzymes. Once we obtain the optimal sequences that catalyze RNA cleavage, we plan to examine further the role of GlcN6P though use of hyperactivated substrates and compare the cleavage mechanism of the GlcN6P-dependent DNAzyme to that of the natural glmS ribozyme.
Keywords: SELEX, Ribozyme, 5-s-phosphorothiolate
Abstract:
Riboswitches are a type of natural genetic control element that use an untranslated sequence of mRNA to form a binding pocket for a metabolite that regulates expression of that gene. Thus, an mRNA that contains a riboswitch is directly involved in regulating its own activity, depending on the presence or absence of its target molecule. The ykkCD riboswitch, discovered in Bacillus subtilis, is an ideal target to tackle the growing problem of antibiotic resistance by pathogenic bacteria. We propose that it regulates the expression of a multidrug-resistant efflux pump. Our goal is to decipher the molecular mechanism by which the ykkCD riboswitch triggers the expression of the multidrug-resistant efflux pump. Fluorescent binding assays will be used to show that the ykkCD riboswitch aptamer domain specifically recognizes what we hypothesize to be aromatic cations as ligands.
Keywords: riboswitch, fluorescence, binding
Abstract:
Find RNA 3D (FR3D), is a suite of programs for finding motifs in RNA atomic-resolution 3D structures. A base-centered approach was implemented to construct efficient, yet exhaustive motif search procedures using geometric, symbolic, or mixed representations of RNA structure. For geometric searches, a user-supplied 3D Query Motif is used to find and score geometrically similar candidate motifs, without regard to nucleotide identity or the sequential position of their nucleotides in the RNA chain or chains. To score and rank candidate motifs, FR3D calculates a geometric discrepancy by first rigidly rotating candidate motifs to optimally align with the Query Motif and then comparing the relative orientations of the corresponding bases in the query and candidate motifs. Candidate motifs may be screened using symbolic constraints to identify those that contain particular bases, basepair types, base-stacking arrangements, base-phosphate interactions, or that conform to sequence continuity constraints. Purely symbolic searches for motifs are also implemented. The Display module provides tools to view, align, cluster and calculate an exemplar for the candidate motifs. It also allows one to superimpose motifs and display neighborhoods. Searches can be saved and written out to PDB files. FR3D is an open source software that has been developed and tested under Windows XP/Mac OS X 10.4, and is capable of running on any PC or Mac platform with Matlab 7.4 or higher installed version. A stand-alone version is also available for the PC platform. FR3D can be downloaded at: http://rna.bgsu.edu/FR3D.
Keywords: RNA 3D Motif, Geometric Search, Interactions
Abstract:
Sarcin-like ribotoxins comprise a family of site-specific endoribonucleases that are conserved in both structure and function. Of all the ribosomal phosphodiester bonds, the ribotoxins cleave only one, in the conserved sarcin/ricin loop that folds into a tetraloop and bulged-G motif. Cleavage of a single bond in the tetraloop inhibits translation and ultimately triggers apoptotic cell death. Previously, we showed that cleavage by the sarcin–like ribotoxin restrictocin occurs via two steps: formation of a nonspecific electrostatic complex (E:S) followed by site-recognition and cleavage (NSMB 13, 436 and Biochemistry 46, 12744). Here, we kinetically analyze a series of interface mutants to identify residues that contribute to each step. To identify which residues contribute to binding in the E:S complex we determined the salt dependence of each mutant. Relative to the wild-type salt dependence, only subset of the mutants clustering near but outside of the active site shows significant changes–K110A, K111A and K113A (the lysine triad). Interestingly, the lysine triad also contributes to the subsequent site recognition step. Next, we identified residues that contribute to the 1,000-fold specificity exhibited by restrictocin for the SRL over other substrates. Unexpectedly, mutation of active site residues Y47 and H49, which are expected to interact equally well with any RNA sequence, had no effect on non-specific cleavage but abolished specific cleavage, indicating a role for the active site in specificity. Similarly, mutation of residues forming substrate specific contacts also reduces specific cleavage with little to no effect on non-specific cleavage. Our findings provide support for a model in which electrostatic contacts by the lysine triad position the active site face toward the substrate. Subsequently, the lysine triad contacts the bulged-G motif and active site residues contact the tetraloop of the SRL. Only formation of both sets of contacts permit proper docking of this substrate in the active site to fully engage Y47 and H49 and thereby enhance specific cleavage by 1,000-fold relative to non-specific cleavage.
Keywords: sarcinricin loop, RNA-protein recognition, ribonuclease
Abstract not available online - please check the printed booklet.
Abstract:
The function of large non-protein-coding RNAs (ncRNA) has remained largely unknown, but they are thought to play important roles in generating the complexity seen in higher eukaryotes. We are interested in defining the rules that govern the mechanism of function of large non-coding RNAs, since despite a number of recent studies, very little is known about the mechanistic aspects of their function. In order to tackle this question, we created a database of more than one hundred large ncRNAs for which at least a general function has been described. Using bioinformatics analysis tools, we searched for sequence elements in the ncRNAs which might be important for their function, such as the presence of transposable elements, small opening reading frames, microRNA precursors, and the levels of conservation. We also determined if any of these RNAs overlapped in sense or antisense direaction with any known genes. Strikingly, over 20% of the transcripts described as large ncRNAs in the database contain hairpins identical to known miRNA precursors in their exons or introns. In some cases, alternatively splicing leads to removal or inclusion of a miRNA precursor sequence. Interestingly, some ncRNAs contain different miRNA precursors for different alternatively spliced isoforms. About 70% of ncRNAs in the database contain transposable elements (TE). While for most ncRNAs, TEs amount to 10-50% of their sequence, for some the share of TEs can reach as high as 70%, consistent with the rapid evolution of this class of transcripts. About 46% of the ncRNAs contain one or more opening reading frames of greater than 300 nucleotides. Close to 30% of the studied ncRNAs were at least partially antisense to a known transcript. Work is underway to further analyze the various features of known ncRNAs. Ultimately, the insights gained from this study will lead to the development of a functional classification system for these mysterious, novel class of cellular regulators.
Keywords: non-coding RNAs
Abstract:
Nucleic acid chaperone proteins facilitate DNA and RNA rearrangements resulting in a thermodynamically more stable conformation. In retroviruses, the small, highly basic nucleocapsid (NC) protein serves as a chaperone and facilitates many nucleic acid restructuring events in the retroviral life cycle. Feline immunodeficiency virus (FIV) is a retrovirus that naturally occurs in the domestic cat and produces symptoms similar to those found in HIV-infected humans. Compared to HIV, little is known about the molecular determinants of FIV NC chaperone activity. In this work, we carry out biochemical and biophysical studies to investigate FIV NC's chaperone function. Sedimentation assays were used to characterize NC-facilitated nucleic acid aggregation, gel mobility-shift assays have measured NC-guided annealing of DNA/RNA, and fluorescence anisotropy experiments provided apparent Kd values for FIV NC binding to DNA and RNA constructs. These results are compared to biophysical data obtained from experiments using HIV-1 NC and HTLV-1 NC. Future experiments will feature mutational analysis of FIV NC followed by characterization in vitro and in cell culture.
Keywords: retrovirus, chaperone, nucleocapsid
Abstract:
Riboswitches are highly structured mRNA elements that specifically bind to small metabolites to regulate gene expression in a cis-fashion. Research in our lab focuses on testing whether the ykkCD putative riboswitch acts as a toxin sensor to trigger expression of a multidrug resistance efflux pump (also called ykkCD) in response to toxic compounds. Efflux pumps are membrane transporters involved in the extrusion of toxic substrates from the cells into the external environment thereby playing an important role in bacterial defense against antibiotics. In this work we tested whether expression of the ykkCD efflux pump is up-regulated or down-regulated in response to toxic compounds. We used the Gramm-positive model organism Bacillus Subtilis for our studies. Cells were grown in the presence of various antibiotics and ykkCD efflux pump mRNA levels were detected using Nucleic Acid Sequence Based Amplification (NASBA). NASBA is a very sensitive technique and ideal to detect small changes in mRNA levels. Our work shows that expression of the ykkCD multidrug-resistance efflux pump is upregulated in tetracyclin but not in streptomycin or chloramphenicol treated cells. Since bacterial strains are showing increased resistance to various drugs it is very important to uncover how bacterial defense is triggered in response to antibiotics. We posit that the ykkCD putative riboswitch could serve as a target in the fight against bacterial pathogens.
Keywords: riboswitch, gene expression regulation, qPCR
Abstract:
Addition of a single G-1 to the 5' end of tRNAHis, in the 3'-5' direction, is an essential and universally conserved modification. This activity is required for recognition of tRNAHis by HisRS and subsequent aminoacylation. The enzyme that catalyzes this reaction, tRNAHis guanylyltransferase (Thg1), was first identified in S.cerevisiae.
G-1 addition in prokarya differs from eukarya as it is Thg1 independent because G-1 is genomically encoded. Nevertheless, protein sequences homologous to yeast Thg1 are found in several bacterial organisms. Since a role for Thg1 in tRNAHis maturation is not predicted in bacteria, function of these homologs is unclear. To address this, we have cloned B.thuringiensis Thg1 (BtThg1) to compare its biochemical activities to those of yeast Thg1. We determined that BtThg1 can catalyze G-1 addition but exhibits differences from yeast Thg1. To quantify these differences, we measured steady state kinetic parameters with ppptRNAHis and ppptRNAPhe, which have already been characterized as yeast Thg1 substrates. BtThg1 exhibits KM for tRNAHis similar to KM for tRNAPhe indicating a lack of selective recognition of tRNAHis over other tRNA species, consistent with lack of involvement of Thg1 in tRNAHis processing in bacteria. These data imply a possibility of a hitherto unknown function associated with Thg1.
A second major goal of our research is structural characterization of Thg1 for which no structural information exists. This investigation will provide key insights into mechanisms of Thg1 substrate recognition, binding and catalysis at a molecular level. BtThg1 has been promising in this aspect. We have been able to crystallize the protein and efforts are underway to generate diffraction data and solve its crystal structure. We expect that structural characterization will reveal a multimeric Thg1 enzyme, since AUC sedimentation velocity analysis indicates that BtThg1 exists as a tetramer in solution. This is the first physical demonstration of a multimeric state of Thg1 and further investigation into the involvement of quaternary structure in activity is ongoing.
Keywords: Thg1, tRNAHis
Abstract not available online - please check the printed booklet.
Abstract:
Eukaryotic ribosome assembly is a highly intricate and complex process that includes modification and folding of the 4 ribosomal RNAs (rRNA) as well as binding of the 78 ribosomal proteins. Ribosome assembly requires the action of more than 170 protein factors. [1] Despite the fact that these proteins are highly conserved within eukaryotes and essential for life, their function remains poorly understood. In the yeast Saccharomyces cerevisiae, a protein called Nob1 is required for cell viability. Depletion of this protein leads to a defect in 40S, or small subunit processing. [2] Specifically, it results in accumulation of 20S rRNA, the precursor to 18S rRNA, which is the primary rRNA component of the small subunit in yeast. Because Nob1 contains a PIN domain, it is believed to be the endonuclease responsible for the D-site cleavage to produce the mature 18S rRNA. [3] In addition to the PIN domain, Nob1 also contains a zinc-finger domain, which is a well-known RNA binding domain. Our lab has successfully produced multiple point mutations in these two domains to test the in vivo and in vitro effects of deleting these specific residues. These results, in conjunction with the wild-type data, will provide a clear insight into the function of this putative ribosomal protein factor.
References:
[1] Fromont-Racine, M.; Senger, B.; Saveanu, C.; Fasiolo, F. Gene 2003, 313, 17-42.
[2] Fatica, A.; Oeffinger, M.; Dlakic, M.; Tollervey, D. Mol. Cell. Biol. 2003, 23, 1798-1807.
[3] Fatica, A.; Tollervey, D.; Dlakic, M. RNA 2004, 10, 1698-1701.
Keywords: ribosome, small subunit
Abstract:
Genomic-scale analyses have revealed numerous functional duplications within the canonical translation machinery, such as the occurrence of unrelated class I and II lysyl-tRNA synthetases (LysRS), which can act in concert to aminoacylate non-canonical tRNAs. To date, LysRS1 and LysRS2 have only been found together in the Methanosarcinaceae in the archaea and in a few Bacilli among the bacteria, including Bacillus cereus. The role the two LysRSs play in B. cereus was investigated. It was found that the class I and II LysRSs could act together to aminoacylate a small RNA of unknown function (tRNAOther). tRNAOther is encoded within a pathogenicity island together with several putative virulence factors. In vivo analyses revealed that LysRS1 and tRNAOther were mainly produced during stationary phase. To investigate the role of tRNAOther in B. cereus, we deleted the corresponding gene and screened for changes in growth phenotype compared to wild-type. Deletion of tRNAOther was not deleterious, and growth in rich media and ability to sporulate were virtually unchanged. Changes were observed in stationary phase such as de-regulation of the production of a bacteriocin-like inhibitory susbstance, and increased responsiveness to various germinants. Extensive screening for growth phenotype changes (~1900 conditions) was carried out by performing phenotypic microarray analyses (BIOLOG, Inc.). These studies revealed significant changes; B. cereus ÄtRNAOther gained resistance to several antibiotics (eg., erythromycin and oleandomycin), but also acquired sensitivity to a number of compounds including antibiotics, cationic detergents and positively charged ionophores. tRNAOther could not be detected in polysomes isolated from cells collected during exponential or early stationary phases, indicating a function beyond translation.
Keywords: Aminoacyl-tRNA synthetases
Abstract:
The goal of this research is to identify the post-transcriptional and post-translational modifications of ribosomal subunits produced from the presence of the antibiotic, erythromycin. Mass spectrometry (MS) based methods are applied to examine ribosome assembly defects in this research.
Escherichia coli is being used as the initial model system.
Four strains of E. coli K-12 (wild type, N281, N282, SK5665) are grown in the presence and absence of erythromycin at 27oC and 37oC. Liquid chromatography with ultraviolet absorption (LC-UV) and mass spectrometry (LC-MS) are used to identify post-transcriptional modifications arising in ribosomal RNA (rRNA) found by comparing treated cells to untreated cells grown at the same temperature. The ribosomal proteins of erythromycin treated cells are then compared to ribosomal proteins of erythromycin absent cells grown at the same temperature using matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS).
Stalled intermediates of strain SK5665 grown with erythromycin at 27oC have been compared to cells grown without the drug at 27oC, with and without erythromycin at 37oC, as well as to K12 cells grown with and without the drug at both temperatures. Stalled intermediate precursors result from 50S assembly inhibition by erythromycin. Current data suggests that there may be a lack of proteins in this stalled intermediate which is owed to an inability of the proteins to bind to the 23S rRNA in the correct order. The inability for proteins to bind may be due to missing a post-transcriptional modification(s) in the 23S rRNA, leading to misfolding of the 23S rRNA.
References:
1. Champney, W. S. Chittum, H. S. Journal of Bacteriology 1994, 176, 6192-6198.
2. Champney, W. S. Current Topics in Medicinal Chemistry 2003, 3, 929-947.
3. Crain, P. Methods in Enzymology, 1993, 193, 782-791.
Keywords: Nucleosides, High Pressure Liquid Chromatography (HPLC), Mass Spectrometry
Abstract not available online - please check the printed booklet.
Abstract:
Estrogens are steroid hormones that are key regulators of a wide variety of biological processes including growth, development and reproduction. Biological effects of estrogen are mediated through estrogen receptor &alpha and &beta, members of the nuclear receptor family of transcriptional activators (1). While there has been a tremendous progress in deciphering transcriptional mechanisms of estrogen action less is known about its overall impact on mRNA decay even though this comprises one of the major mechanisms regulating gene expression.
In Xenopus hepatocytes estrogen activates a polysome-associated endonuclease, PMR1 that catalyzes the coordinate destabilization of the serum protein mRNAs (2). We previously showed that PMR1 forms a specific complex with its translating target mRNAs, is activated to initiate mRNA decay and also identified that tyrosine phosphorylation by c-Src kinase is involved in allowing PMR1 to bind to polysomes bearing its translating substrate mRNA (3,4). However the mechanism that activates polysome bound PMR1 is not known. This project seeks to characterize the ER activation of PMR1 mediated mRNA decay, probe into the phosphorylation events and link these to the signal transduction pathway[s] activated by between estrogen binding to its receptor. Importantly, we identified an LXXLL nuclear receptor-binding motif in PMR1 and found that ER&alpha and PMR1 interact in vivo. Interestingly we also found that this interaction is modulated by E2 in a time dependent manner. We know from previous work that binding to polysomes is not enough to activate PMR1-mediated mRNA decay, and the recovery of ER&alpha with PMR1 raises the novel possibility that ligand-bound receptor activates mRNA decay by binding directly to the effector mRNA endonuclease.
The central hypothesis of this proposal is that estrogen causes ER&alpha to bind to the PMR1-substrate complex, thus activating the decay of a selective set of growth-regulatory mRNAs. There is no precedent for this in literature and it has opened a whole new direction of possibilities involved in the signal transduction events associated with E2 induced PMR1 mediated mRNA decay.
References:
1. Deroo BJ, Korach KS. (2006) Estrogen receptors and human disease. J Clin Invest.116, 561-70.
2. Dompenciel RE, Garnepudi VR and Schoenberg DR. (1995) Purification and characterization of an estrogen-regulated Xenopus liver polysomal nuclease involved in the selective destabilization of albumin mRNA. J. Biol. Chem. 270, 6108–6118.
3. Yang F, Schoenberg DR. (2004). Endonuclease-mediated mRNA decay involves the selective targeting of PMR1 to polyribosome-bound substrate mRNA. Mol Cell. 14, 435-45.
4. Peng Y, Schoenberg DR. (2007). c-Src activates endonuclease-mediated mRNA decay. Mol Cell. 25, 779-87.
Keywords: Estrogen receptor, PMR1, mRNA decay
Abstract:
To satisfy the high demand for ribosome synthesis in rapidly growing eukaryotic cells, short duplexes between the U3 small nucleolar RNA (snoRNA) and the precursor ribosomal RNA (pre-rRNA) must form quickly and with high yield(1-3). These interactions, designated the U3-ETS and U3-18S duplexes, are essential to initiate the processing of small subunit rRNA. Previously, we developed assays designed to mimic these RNA-RNA interactions and showed qualitatively that these duplexes form only after addition of one of two proteins: Imp3p or Imp4p(4). Here, we developed fluorescence-based assays to determine whether these proteins assemble and possess RNA chaperone activity sufficient to satisfy the high U3-ETS duplex yield and rapid U3-18S duplex formation (t1/2 ~ 85 sec) expected in vivo. In the absence of protein, the U3-ETS duplex is weak due to short duplex length. A kinetic barrier limits formation of the U3-18S duplex because the U3-stem structure must unfold to expose its base-pairing site. Both proteins assemble with the U3 snoRNA into a chaperone complex, which is the smallest relevant complex to ensure sufficient U3-pre-rRNA duplex formation and yield. This complex stabilizes the U3-ETS duplex Kd by two orders of magnitude with a negligible effect on the duplex kon. The chaperone complex also stimulates U3-18S duplex formation presumably opens up the U3-stem structure to expose the 18S base-pairing site. Our findings demonstrate that formation of the U3-18S duplex will occur too slowly (t1/2 >> 85 sec) in the absence of protein for any concentration of pre-rRNA. In contrast to the absence of protein, assembly of the chaperone complex sufficiently accelerates U3-18S duplex formation to satisfy the need for rapid formation of this duplex under a wide range of pre-rRNA concentrations. The activities of this chaperone complex result in high U3-ETS duplex yield and a fast formation of the U3-18S duplex, thereby ensuring that the U3-pre-rRNA interactions limit neither ribosome biogenesis nor rapid cell growth.
References:
References
(1) Beltrame, M.; Tollervey, D. EMBO J 1995, 14, 4350-6.
(2) Sharma, K.; Tollervey, D. Mol Cell Biol 1999, 19, 6012-9.
(3) Osheim, Y. N.; French, S. L.; Keck, K. M.; Champion, E. A.; Spasov, K.; Dragon, F.; Baserga, S. J.; Beyer, A. L. Molecular Cell 2004, 16, 943-54.
(4) Gerczei, T.; Correll, C. C. Proc Natl Acad Sci U S A 2004, 101, 15301-6.
Keywords: chaperone, conformational change, ribosome biogenesis
Abstract:
In mammalian cells, pre-mRNA splicing factors and other pre-mRNA processing factors are stored in nuclear bodies called nuclear speckles. Proteomic analysis of nuclear speckles reflected enrichment of splicing factors in nuclear speckles and revealed 33 novel speckle proteins including Son1. Son is a large protein (2564 amino acid length) that contains six types of repetitive sequences that cover approximately one-third of its sequence and that are not found in any other protein. This study aims to determine nuclear functions for Son in pre-mRNA transcription and splicing as well as in structural organization of speckles.
We have recently generated rabbit polyclonal antibodies against both the amino- and the carboxy-termini of Son. Immunofluorescence localization of endogenous Son with these antibodies shows colocalization of Son with splicing factor 2/alternative splicing factor (SF2/ASF) in nuclear speckles. In addition, we fused Son with yellow fluorescent protein (YFP) and stably expressed YFP-Son in Hela cells where it also co-localizes with SF2/ASF in nuclear speckles. Interestingly, Hela cells that are treated with Son siRNA oligos show a complete reorganization of nuclear speckle components (including SR proteins and snRNP proteins) from their typical irregular shape into a donut-like shape. Son depleted cells also show a hampered cell growth pattern and an accumulation in G2/M phase of the cell cycle. The altered organization of nuclear speckles and an impeded cell growth pattern observed after Son depletion implicates Son in a novel and essential role in the structural maintenance of nuclear speckles and in cell cycle progression.
References:
1. Saitoh, N., C. S. Spahr, S. Patterson, P. Bubulya, A. F. Neuwald and D. L. Spector. 2004. Proteomic analysis of interchromatin granule clusters. Mol. Biol. Cell. 15:3876-3890
Keywords: nuclear speckles, cell cycle
Abstract:
The 3’-end of a messenger RNA is generally defined by a poly(A) tail. The position of a poly(A) site of an eukaryotic mRNA is determined by sequence signals in the precursor-mRNA. Alternative polyadenylation (APA), in which a poly(A) tail is located at a different location of individual transcripts, allows the same DNA template to produce different isoforms of mRNA. To study poly(A) signals and APA in rice and a model algal species, Chlamydomonas reinhardtii, at a genome level, we constructed two datasets of 55,742 and 16,952 authenticated unique poly(A) sites, respectively. In rice, we identified the poly(A) signals as the typical tripartite cis-elements, including Far-Upstream-Elements (FUE), Near-Upstream-Elements (NUE) and Cleavage Elements (CE) as previously observed in Arabidopsis. When mapped to the genome, however, 15% of these poly(A) sites were found located in the currently annotated intergenic regions. Moreover, an extensive alternative polyadenylation profile was evident where 50% of the genes analyzed had more than one unique poly(A) site. About 4% of analyzed rice genes possessed alternative poly(A) sites at their introns, 5’-UTRs, or protein coding regions. Studies on the poly(A) signals in Chlamydomonas found a unique set of polyadenylation signals that are distinct from that of other organisms. In contrast to the high-AU content in 3’-UTR of rice, Chlamydomonas shows a high-Guanylate content that transits to high-Cytidylate around the poly(A) site. A dominant poly(A) signal, UGUAA, was found in over 50% of NUEs, and its occurrence may be positively correlated with higher gene expression levels. We also observed a high-level APA in the Chlamydomonas draft genome, with a range up to ~33% genes having at least two unique poly(A) sites, and a range of 1% to 11% (depending upon the stringency of the criteria) of the poly(A) sites residing in introns, 5’-UTRs, or protein coding regions. In conclusion, we identified two distinct sets of poly(A) signals from two evolutionarily distant organisms. However, in either case, alternative polyadenylation are found to be significant, which could play an important role in gene expression regulation.
Keywords: Polyadenylaion signal, Alternative polyadenylation, rice Chlamydomonas
Abstract:
Despite rigorous research in the field of prostate cancer, the molecular mechanisms underlying the critical transition of prostate cancer from androgen-dependent to the fatal androgen-independent stage are poorly understood. Given the important regulatory roles of microRNAs (miRNAs) in carcinogenesis, we asked whether miRNAs are involved in the pathogenesis of androgen-independent prostate cancer. In order to answer this question, we have profiled the expression of 12 miRNAs organized in three clusters (miRNA 17-92 cluster, miRNA 106b-25 cluster and miRNA 23b-24 cluster) in hormone-dependent and hormone-independent prostate cancer cell culture models. About one third of human miRNAs are hosted in the introns of annotated mRNAs. The biological significance of locating miRNAs in the introns of host genes is poorly understood. The prevailing view is that intronic miRNAs are processed from the same primary transcript as their host genes and thus, their expression is regulated by the expression of the host mRNA. However, this hypothesis has not been extensively tested. In this context, we also profiled the expression of the host transcripts – C13orf25, MCM7 and Aminopeptidase O, which harbor the miRNA clusters, 17-92, 106b-25 and 23b-24 respectively. Our data show no significant correlation between the expression profile of the three miRNA clusters and the hormone responsive status of prostate cancer cells. Hence, these miRNAs may not be important for the development of androgen independence. Further, we found no correlation between the expression of host transcript and the expression of its resident miRNAs. This observation raises interesting questions regarding the biogenesis of intronic miRNAs and suggests the possibility of independent transcription and/or posttranscriptional regulation of intronic miRNAs.
Keywords: Prostate cancer, microRNA, host transcripts
Abstract not available online - please check the printed booklet.
Abstract:
Regulatory micro (mi) RNAs are evolutionarily conserved large class of RNAs molecules of 19-24 nucleotides in length and are found in various organisms including humans. These novel noncoding RNAs regulate gene expression by targeting the 3’ untranslated region (UTR) of mRNAs. A large number of miRNA genes are encoded in mammalian introns. Using bioinformatics approaches we have identified a variety of sequence elements that are found in miRNA neighboring exons and introns of human, mouse, and rat. The identification and functional evaluation of these regulatory motifs and elements would help us to understand their roles in coordinated events of nuclear pre-mRNA splicing and miRNA processing.
Keywords: miRNA, pre-mRNA splicing, Bioinformatics
Abstract:
tRNAHis guanylyltransferase (Thg1) is an essential enzyme in yeast that catalyzes incorporation of a single guanosine residue at the -1 position (G-1) of tRNAHis. G-1 serves as a necessary recognition element for histidyl-tRNA synthetase and is conserved among all tRNAHis species. Thg1 is the only known enzyme that adds nucleotides in the 3'-5' direction and shares no identifiable sequence homology to any other known enzyme, thus its molecular mechanism is unknown.
The first step in G-1 addition involves activation of the 5' end of monophosphorylated tRNAHis by formation of an adenylated intermediate. In subsequent steps the 3'-OH of GTP attacks the intermediate yielding AMP and resulting in the addition of a single GTP to the 5' end of the tRNA. In the final step Thg1 removes pyrophosphate from the G-1 residue yielding mature G-1-containing tRNAHis. A complete understanding of this complex reaction mechanism requires isolation and characterization of each of these catalytic steps individually. Previous studies suggest that adenylation is a major specificity determining step since Thg1 is unable to adenylate tRNA substrates lacking the tRNAHis anticodon (GUG). Therefore, we have first focused on the kinetic characterization of the adenylation reaction. We used transient kinetic techniques (single turnover assays) to explicitly measure the first-order rate constant of adenylation (kaden) of tRNAHis for wild type yeast Thg1. We measured a kaden of 0.25 s-1, which is significantly faster than the previously measured kcat (0.012 s-1), suggesting that adenylation is not the rate determining step for G-1 addition under these conditions. Our results suggest a rapid equilibrium mechanism for adenylation of tRNAHis (k-1 >> kaden), with KD app~ 0.88 µM. To identify catalytic residues that participate in adenylation, we are currently using site directed mutagenesis to identify Thg1 variants that exhibit compromised adenylation activity however still retain that ability to carry out guanylyltransfer.
Keywords: tRNAHis, Thg1
Abstract:
Aminoacyl-tRNA synthetases are essential enzymes that help to ensure the fidelity of protein translation by accurately aminoacylating (or “charging”) specific tRNA substrates with cognate amino acids. Many synthetases have an additional catalytic activity to confer amino acid editing or proofreading. For example, prolyl-tRNA synthetase (ProRS) mis-activates alanine and deacylates mischarged Ala-tRNAPro using an editing active site that is distinct from the site of amino acid activation. A free-standing protein (YbaK) with homology to the ProRS editing domain is present in most bacteria. YbaK has been shown to possess hydrolytic editing activity against mischarged Cys-tRNAPro. However, relatively little is known about the catalytic mechanism of YbaK or other free-standing editing domains at the molecular level. Previously, we demonstrated that the strictly conserved K46 residue in the putative substrate-binding pocket is critical for Cys-tRNAPro editing activity, and that the specificity of trans-editing by YbaK is ensured through formation of a novel ProRS/YbaK/tRNA ternary complex.
To further characterize the trans-editing mechanism of YbaK, we performed extensive Ala-scanning mutagenesis of conserved residues in ProRS and YbaK families, as well as residues identified from substrate docking studies and molecular dynamics simulations. Additionally, to probe the function of the tRNA hydroxyl groups in substrate recognition and catalysis, we tested YbaK activity using dA76 variants of Cys-tRNA. Taken together, the results of these studies allow us to propose a mechanism for Cys-tRNAPro editing by YbaK involving stabilization of substrate functional groups by conserved residues within the substrate-binding site and cysteine-thiolactone formation.
Keywords: Aminoacyl-tRNA synthetase, YbaK, editing
Abstract:
RNAs from all three domains of life—eukarya, bacteria, and archaea—can act as substrates for numerous posttranscriptional modifications, including tRNA editing. Of particular interest is the deamination of adenosine to inosine that occurs at the first or “wobble” position of the anticodon in tRNAs. Given the ability of inosine to base pair with cytosine, uridine and adenosine, this editing event allows a single tRNA to decode multiple codons. In eukaryotes, adenosine deaminases acting on tRNA (ADATs) are heterodimeric enzymes responsible for conversion of adenosine to inosine. Of the two subunits, ADAT2 is known to be catalytic while the contributions of ADAT3 to catalysis, substrate recognition and binding are unknown. Here we show, through systematic mutations of the ADAT2/3 enzyme from Trypanosoma brucei, the role that both the ADAT2 and ADAT3 subunits play on editing activity. Single amino acid substitutions to the proposed active and "pseudo active" sites of these subunits, show that as expected ADAT2 plays a prominent role in catalysis, but importantly, suggests that ADAT3 also plays a catalytic role. We also show that deletion of a run of lysine and arginine residues at the c-terminus of ADAT2 impairs tRNA binding, defining a potential tRNA binding site on the enzyme that is distant from the active site. This observation provides a first clue as to the basis for substrate recognition for this group of eukaryotic enzymes and has implications for the evolution of new substrate specificities among eukaryotic deaminases.
Keywords: tRNA editing, Adenosine deaminase
Abstract:
Riboswtiches are recently discovered modes of RNA dependent gene regulation of gene expression through allosteric structural changes triggered by the binding of a small molecule. Bacteria have two ways of potential rendering antibiotics inactive, they can either (1) degrade the antibiotic, or (2) with the help of efflux pumps, they can export antibiotics from the bacterial cell. We hypothesize that the ykkCD riboswitch regulates expression of this efflux pump, and works as a potential toxin sensor by binding to antibiotics. With this, the ykkCD riboswitch could serve as a target in the fight against antibiotic resistance. We will present initial characterization of the previously uncharacterized ykkCD riboswitch. I will test how the structure of the riboswitch changes upon antibiotic binding by mapping the conformation of the OFF and the ON state of the ykkCD riboswitch via footprinting.
Keywords: Riboswitch, gene regulation, footprinting
Abstract:
In Saccharomyces cerevisiae, the final cytoplasmic step in the maturation of 40S ribosomes involves cleavage of 20S rRNA to generate the mature 3´ end of 18S rRNA. Several accessory factors are involved in this step, mutations of which lead to accumulation of 20S rRNA in the cytoplasm and failure to produce mature, active ribosomes (1). One such factor is Fap7 an essential protein with ATPase sequence homology believed to act through transient association with the maturing small subunit (2). Conserved amino acids predicted to be involved in ATP hydrolysis are required for Fap7's 18S rRNA processing activity (2). We have demonstrated that Fap7 has ATPase activity. Using a combination of kinetic, fluorescence and proteolysis experiments we have demonstrated that Fap7 dimerizes in the presence of ATP forming an asymmetric dimer. Once both ATP binding sites are filled, ATP is hydrolyzed in a sequential manner. We are currently investigating how this ATPase activity might be used during ribosome assembly and are specifically testing the hypothesis that ATP hydrolysis by Fap7 is used for remodeling of pre-ribosomes. Interestingly, Fap7 has also been implicated in a separate but not necessarily unrelated role, regulation of the Skn7/Pos9 transcriptional response to oxidative stress (3). Fap7 may thus integrate transcriptional and translational response to oxidative stress.
References:
1. Peng et al. Cell. 2003 113(7): 919-33.
2. Granneman et al. Mol Cell Biol. 2005 25(23): 10352-64.
3. Juhnke et al. Mol Microbiol. 2000 35(4): 936-48.
4. Zemp and Kutay. FEBS Letters. 2007 15: 2783-2793.
Keywords: rRNA processing, ribosome assembly, yeast
Abstract:
c-Myc is a transcription factor that regulates numerous processes including cell cycle, apoptosis, cellular differentiation, cellular metabolism and genomic instability. In normal cells, expression of c-Myc is tightly regulated by external signals such as growth factors and extracellular matrix. However in stressed and cancerous cells, c-Myc is generally over-expressed by chromosomal translocation, gene amplification, or stabilization of its mRNA. We hypothesize that MYC is a target of microRNA (miRNA). miRNAs are 20-22bp short RNAs that regulate gene expression by binding target mRNA 3’-UTR, leading to mRNA degradation or translational repression. We used a reporter construct carrying c-Myc binding sites to screen miRNAs targeting MYC. The results demonstrated that miR-212 (30%), miR-203 (40%), miR-33b (50%) and miR-33a (50%) down-regulated reporter activity. Next, we cloned the 3’-UTR of MYC into a reporter vector and performed assays in cell transfected with miRNAs. We found that miR-33a (60%), miR-33b (50%), miR-203 (40%) down-regulated the expression of the reporter gene located upstream of the MYC 3’-UTR. Finally, we performed colony formation assay with rat RK3E-cmyc cells, which constitutively express c-Myc. Our results showed that miR-212 (85%), miR-33a (50%), miR-33b (60%) and miR-203 (17%) reduced colony formation. We will continue to investigate whether MYC is an authentic target of these miRNAs.
Keywords: MicroRNA, c-Myc, MYC
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Polyadenylation, a step in posttranscriptional processing, is a key part of the formation of most eukaryotic mRNAs. Poly(A) signals in mammals and yeast are fairly well understood, but much about the signals and the proteins that interact with them is not known in plants. In an attempt to better understand poly(A) signaling in plants, we used a novel program, Signal Sleuth, to analyze the poly(A) signals in genes isolated from a previously characterized, poly(A) site aligned data set (8K dataset). The genes isolated from the 8k represented either the pollen transcriptome or seed storage protein genes. The objectives of examining these two gene sets were to find unique signals, a pronounced signaling preference, or new signaling elements. Our analysis gives us new insights into the signal specificity of the plant poly(A) apparatus, and suggests the methods we should use to further our knowledge of the proteins in the poly(A) complex.
Keywords: polyadenylation, signaling, processing
Abstract:
Bacterial manganese superoxide dismutase (MnSOD) has been shown to localize to the chromosomal portion of the cell and impart protection from ionizing radiation to DNA. The binding affinity of bacterial MnSOD to non-sequence specific double stranded oligomeric DNA has been quantitated previously by nitrocellulose filter binding and gel shift assays. In our lab, using fluorescence quenching titrations, we have found that polynucleotides bind to MnSOD in the following affinity hierarchy, poly(dU)~poly(U) > dsDNA~poly(A) > poly(C). The current study attempts to verify these observations using DNAse protection assays using 5’-biotin labeled homooligonucleotides. The composition of the oligonucleotides used was C20T20, T20C20, A20C20, C20A20, A20G20, and G20A20. Some of these assays confirmed the hierarchy found in previous studies. Some results uncovered previously unknown properties of ExoI nuclease. Contradictory results were also obtained. Further explanations of these contradictory results are being sought.
Keywords: DNAse, specificity, superoxide dismutase
Abstract:
Cotranscriptional cleavage events, driven by specific cis-acting elements known as CoTC, are required downstream of some poly(A) signals to terminate transcription. Little is known about these elements, but in some cases, a pause site can replace the CoTC element. The IgM gene is alternatively processed to produce two mRNA isoforms by either splicing the primary transcript between the Cµ4 and the M1 exons (splice) or cleaving and polyadenylating it at the µs poly(A) signal (pA); the pA/splice RNA expression ratio increases during B cell maturation. A pause site was identified downstream of the µs pA signal, but it is not known whether the transcript also contains a CoTC element. To explore whether a CoTC-like element exists within the IgM gene that may contribute to developmental changes in Ig expression, we analyzed the RNA downstream of the µspA signal by semi-quantitative RT-PCR using sets of primers that span 4 Kb of sequence downstream of the µspA signal. The presence of a CoTC element would be detected as an abrupt decrease in transcript somewhere downstream of the ěspA signal. However, we observed a gradual decrease in transcripts over the region in both B cells and plasma cells, suggesting a natural CoTC element is not present. To examine the effect a CoTC element would have on the competition between the splice and cleavage-polyadenylation reactions, we inserted the B-globin CoTC sequence into two locations downstream of the µspA signal, both in the presence and absence of the pause site. We observed that inclusion of the B-globin CoTC element toward the end of the intron causes cotranscriptional cleavage but does not greatly affect the RNA processing reactions, except as expected due to the size of the insert. However, inserting the B-globin CoTC element close to the µspA site not only caused cleavage and early termination of transcription, but also caused an increase in the pA/splice ratio. This suggests there is a position effect of the inserted CoTC element on the competing polyadenylation and splicing reactions within the IgM transcripts.
Keywords: Co-transcriptional Cleavage, IgM, RNA processing
Abstract:
Abstract:
Nuclear speckles are nuclear storage and assembly sites for pre-mRNA processing factors. Btf (Bcl-2-like transcription factor or BCLAF) was identified in a proteomic analysis of purified nuclear speckles and it localized in a unique pattern that resembles transcription sites(1). Our hypothesis is that Btf has a role in the coordination of transcription and co-transcriptional processing of pre-mRNAs. Recently reported cap-dependent association of Btf with in-vitro synthesized affinity purified MS2-AdML-M3 mRNPs is consistent with such a role(2). Additional evidence for Btf in transcription and/or splicing comes from our observations showing that Btf accumulates on a reporter gene locus in situ. Since Btf is enriched on the transcriptionally activated locus, but not on the inactive locus, we hypothesize that Btf is recruited to this locus during gene activation. To test this hypothesis, we are using this reporter locus to compare the timing of Btf recruitment with chromatin decondensation and the accumulation of chromatin remodeling factors, transcription factors and splicing factors.
SR splicing factors are typically targeted to nuclear speckles by RS domains. Although Btf has an arginine-serine-rich (RS) domain, it localizes predominantly around the periphery of nuclear speckles. We have performed deletion analysis to understand the unique Btf localization pattern. We have confirmed that the RS domain of Btf can function as a speckle targeting sequence on its own; however, removal of the Btf RS domain does not significantly change the Btf localization pattern. This suggests that Btf localization may be regulated differently than other speckle proteins. Our deletion analysis indicates that a specific region of Btf prevents its speckle localization, and that removing this region directs Btf to nuclear speckles. Ongoing studies aim to determine if this regulation occurs via post-translational modification of Btf, or by intermolecular interactions with binding partners.
References:
References:
1.Saitoh, N. et al., 2004. Mol. Biol. Cell 15: 3876-3890.
2.Merz, C. et al., 2007. RNA. 13:1-13.
Keywords: splicing, Btf
Abstract:
Alternative splicing of eukaryotic mRNAs increases the number of proteins that are produced from a single gene. Dietary status and individual nutrients are potent regulators of alternative splicing as we observe with glucose-6-phosphate dehydrogenase (G6PD). G6PD expression is regulated by intron retention in which the rate of intron removal and splicing determines the amount of the translated protein. The amount of G6PD mRNA changes in response to nutrient stimuli such that insulin and dietary carbohydrate enhance intron removal, which increases the accumulation of mature mRNA. Starvation and polyunsaturated fatty acids decrease the rate of intron removal, leading to intron retention. These properties make G6PD an excellent model to determine how nutrients regulate pre-mRNA splicing. Exon 12, a constitutive exon of G6PD mRNA, contains an ESS within nucleotides 43-72 that inhibits intron removal. Through RNA affinity chromatography and proteomic approaches, hnRNPs K, L, and A2/B1 were identified as putative regulatory proteins. We hypothesize that these hnRNPs are splicing silencer proteins, as we have observed greater binding of the hnRNPs to the regulatory element during starvation, corresponding to a reduction in splicing, than during re-feeding when splicing is enhanced. The 30 nucleotide regulatory element in exon 12 also contains an ESE. Deletion of the entire element from an in vitro splicing substrate does not restore splicing. Consistent with the regulatory element containing an ESE, binding of SRp20 and SRp75 was detected by RNA affinity chromatography; furthermore, binding of SRp20 is enhanced by re-feeding, corresponding to an increase in splicing. In hepatocytes, insulin stimulates phosphorylation of several SR proteins while addition of arachidonic acid inhibits this effect. Thus, nutrients can regulate the activity of splicing regulatory proteins causing changes in the splicing of G6PD and potentially other pre-mRNAs. We hypothesize that mutually exclusive binding of hnRNPs and SR proteins regulates this model of intron retention. [Supported by DK046897 and a WV Graduate Fellowship in Science, Technology, Engineering and Math.]
Keywords: splicing, nutrients, SR proteins
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
PCF11 and CLP1 interacts with each other and are required for the 3’-end specific cleavage of pre-mRNA in yeast and human. The Arabidopsis genome possesses four PCF11-Similar proteins (PCFS1, PCFS2, PCFS4 and PCFS5) and two CLP1-Similar proteins (CLPS3 and CLPS5). In this study, we provide evidence that PCFS4 directly interacts with CLPS3 and formed complex with other polyadenylation factors in vivo. Intriguingly, in addition to the polyadenylation factors, two flowering time regulators FY and FCA were also found in the complex. FY and FCA interact with each other and are required for regulating the alternative polyadenylation of FCA pre-mRNA, and therefore the flowering time. Consistent with the physical links among PCFS4, CLPS3, FY and FCA, both the loss-of-function mutant pcfs4 and gain-of-function mutant clps3 showed altered flowering time and altered alternative polyadenylation pattern of FCA pre-mRNA. We therefore conclude that the polyadenylation apparatus was recruited to process FCA mRNA, likely through FCA.
Keywords: PCFS4, CLPS3, FCA Alternative polyadenylation
Abstract:
Protein synthesis occurs in several steps including formation of aminoacyl-tRNAs by aminoacyl-tRNA synthetases (aaRSs) which are then delivered to the ribosome by the elongation factor-Tu (EF-Tu) for decoding. It is important to maintain low error rates at each stage in order to achieve overall translational accuracy and thereby normal functioning of cells. AaRSs pair tRNAs with the correct amino acids through substrate specificity and editing of incorrect products. In the PheRS system, PheRS is specific for its substrate phenylalanine (Phe) but it is also known to misactivate tRNAPhe with tyrosine (Tyr). Mischarged Tyr-tRNAPhe is hydrolyzed by a post-transfer editing mechanism. It was thought that any aminoacyl-tRNA released from an aminoacyl-tRNA synthetase would be sequestered by EF-Tu and incorporated into ribosomal protein synthesis. Our biochemical studies instead show that aminoacyl-tRNAs can readily dissociate from their ternary complex with EF-Tu and re-associate with AaRSs. In the case of a mischarged species, this enables the AaRS to rectify its mistake via the editing pathway. Through in vitro protein synthesis assays we also show that resampling of mischarged tRNAs by AaRSs contributes greatly to translational fidelity, providing additional quality control prior to translation elongation.
Keywords: Editing, Quality control, translation
Abstract:
Ribosomes are the machinery responsible for catalyzing protein synthesis in all cells. While the process by which these essential complexes form is well studied and understood in prokaryotes, in eukaryotes the formation is much more complex and therefore not entirely known. What makes the eukaryotic ribosome more complicated is the observation that assembly requires >170 accessory proteins in order to process, fold and bind 78 ribosomal proteins to the 5.8S, 25S and 5S ribosomal RNAs (rRNAs) that make up the large subunit and the 18S rRNA that comprises the small subunit(1). Of these four rRNAs, three (5.8S, 25S and 18S) are co-transcribed in a single transcript known as the 35S rRNA(1). In order for the mature rRNAs to be formed from the 35S rRNA, a series of processes and cleavages must occur. In the maturation of the 18S rRNA, for example, cleavage must occur at the site known as A2, which separates the 18S segment from the rest of the 35S rRNA. We hypothesize that in order for this step to occur, an inhibitory helix formed by base pairs alternative to the mature structure must be disrupted. Our model suggests that Rok1, an ATP-dependent RNA helicase, interacts with the rRNA via Rrp5, an rRNA binding protein, and uses its ATPase activity to unwind this inhibitory duplex. Prior studies have shown that Rok1 is essential for viability and that the depletion of the Rok1 protein blocks synthesis of the 18S rRNA, therefore providing support to the model suggested above(2,3). To prove our hypothesis, we will focus on characterizing Rok1 and its interactions with Rrp5. Thus far, we have exemplified Rok1’s ATPase activity and conducted ATPase inhibition studies that suggest that Rok1 binds ADP more tightly than ATP. In addition, we have shown that Rok1 binds rRNA. Future directions include the addition of Rok1, Rrp5 and rRNA to all assays simultaneously in hopes of developing an in vitro helicase assay that will help us to better understand the maturation of 18S rRNA.
References:
1 Fromont-Racine, M.; Senger, B.; Saveanu, C.; Fasiolo, F. Gene 2003, 313, 17.
2 Song, Y.; Kim, S.; Kim, J. Gene 1995, 166, 151.
3 Venema, J.; Bousquet-Antonelli, C.; Gelugne, J.; Caizergues-Ferrer, M.; Tollervey, D. Mol. Cell. Biol. 1997, 17, 3398.
Keywords: ribosomal assembly, 18S rRNA
Abstract:
The bacteriophage phi29 DNA-packaging motor is the most powerful bio-motor constructed to date. The RNA component, called packaging RNA or pRNA, plays important roles in the DNA packaging process. Six pRNA subunits bind to the distal region of the connector of the procapsid and form a hexamer ring. We are interested in elucidating: 1) interactions between pRNA and other motor components, such as connector and the ATPase gp16; and 2) pRNA motion during the DNA packaging process. To this aim, single-molecule (sm-) and time-resolved (tr-) FRET (fluorescence renascence energy transfer) are conducted to calculate the distances constraints. Sm-FRET is based on the fluorescence intensity emitted by a pair of fluorophores depending on the distance between the donor and the receptor at single molecule level. While tr-FRET depends on the ensemble data instead of single molecule, it can report on photophysical events that are difficult or impossible to observe by fluorescence intensity imaging and has the ability to deconvolute multiple components and get more accurate energy transfer data. Results from these methods are complimentary. Our results indicate that the properties of fluorescent dyes need to be carefully examined so that they can be better suited for the specific experiments. Cyanine dyes, such as Cy3 and Cy5, are good sm-FRET probes for their photo-stability. Stable fluorescence curve with less fluctuation and blinking were obtained. However, the Cy3/Cy5 pair displayed complicated lifetime profiles. Alexa dyes, such as 546 and 647, displayed simple lifetime profiles; however, required different environment to achieve photo-stability and in most cases displayed fluctuated intensity curves in sm-FRET measurement. Though small variation were found, distance constraint data strongly support the global structure of the three D models of phi29 pRNA constructed in this lab (Hoeprich S, Guo P. Computer modeling of three-dimensional structure of DNA-packaging RNA (pRNA) monomer, dimer, and hexamer of Phi29 DNA packaging motor. J Biol Chem. 2002. 277(23):20794-803).
References:
Hoeprich S, Guo P. Computer modeling of three-dimensional structure of DNA-packaging RNA (pRNA) monomer, dimer, and hexamer of Phi29 DNA packaging motor. J Biol Chem. 2002. 277(23):20794-803
Keywords: phi 29, FRET, photostability
Abstract:
The T box transcription antitermination system has been found in many Gram-positive bacteria. This system contains a highly-conserved sequence of nucleotides known as the T box sequence and regulates transcription through a unique interaction between the tRNA and the 5’ leader region of the nascent mRNA. This interaction involves, in part, the base pairing of the tRNA accepter end with four bases of the antiterminator, which prevents the formation of an alternative terminator secondary structure and results in complete transcription of the gene. In an effort to investigate the potential for molecular modulation of this mechanism, a library of small molecules was synthesized and investigated for their binding affinities to the T box antiterminator model AM1A by Fluorescence Resonance Energy Transfer (FRET). The initial screening was conducted by adding the ligands to both a functional and a reduced function FRET-labeled antiterminator model RNA. A subset of compounds was selected for further detailed binding studies. The resulting structure-activity relationship results will be reported.
Keywords: T box tranascription antitermination, Ligand, FRET, structure-activity relationship
Abstract:
T box antiterminator riboswitch regulation is found primarily in Gram-positive bacteria and is a major regulatory mechanism for genes related to amino acid biosynthesis. The 5' untranslated region of these genes monitor and respond to the charging ratio of cognate tRNA. The ultimate objective of this project is to develop novel RNA-targeted medicinal agents through examination of the structure and function of the RNA and associated ligands. A more specific aim is to find peptides that are capable of binding to the antiterminator of the Bacillus subtilis tyrS sequence to disrupt the tRNA-antiterminator complex critical for the functioning of the T box riboswitch. The antiterminator contains the highly conserved T box sequence and a seven nucleotide bulge, of which the first four nucleotides base pair with the tRNA in the complex. The model antiterminator, AM1A, contains the same T box sequence and seven nucleotide bulge and is used in binding studies with various peptide sequences. The goal is to find peptides that will bind well with the antiterminator and then place the ligand complex in further testing to identify the effect of the binding on the tRNA-antiterminator interaction. Fluorescence spectroscopy was used to determine two trends: first, the relative binding affinities of a small library of peptides to the AM1A and second, the effect of the relative position of the amino acids within the peptide on the relative binding affinity.
Keywords: Structure-activity relationship, peptide binding, RNA
