RRM
2006
Rustbelt RNA Meeting
RRM
|
|
|
|
|
Talk abstracts
Abstract:
The SMK box is a conserved riboswitch motif found in the 5’ untranslated region of metK genes (encoding S-adenosylmethionine (SAM) synthetase) in lactic acid bacteria, including Enterococcus, Streptococcus and Lactococcus species. This RNA element binds SAM in vitro, and SAM binding causes a structural rearrangement that sequesters the Shine-Dalgarno (SD) sequence. Mutations that disrupt pairing between the SD and a region complementary to the SD (designated the anti-SD, or ASD) block SAM binding, while compensatory mutations that restore pairing also restore SAM binding. The SMK box sequence from Enterococcus faecalis conferred translational repression of a lacZ reporter when cells were grown under conditions where SAM pools are elevated. Based on these studies, a model was developed in which SAM binding inhibits metK translation by sequestration of the SD region of the mRNA. In the current work, this model was tested using biochemical approaches. Addition of SAM was shown to inhibit binding of 30S ribosomal subunits to SMK box RNA; in contrast, addition of S-adenosylhomocysteine (SAH) had no effect. A mutant RNA defective in SAM binding showed no reduction of ribosome binding in the presence of SAM. Primer extension inhibition assays provided further evidence for SD-ASD pairing in the presence of SAM. These results strongly support the model that SMK box translational repression operates through occlusion of the ribosome binding site.
References:
Fuchs, R.T., Grundy, F.J., and Henkin, T.M. The Smk box is a new SAM-binding RNA for translational regulation of SAM synthetase. Nat. Struct. Mol. Biol. 13, 226-233 (2006).
Keywords: riboswitch, translation, SAM
Abstract:
A number of systems have recently been discovered in which gene expression is regulated through direct sensing of regulatory signals by nascent RNA transcripts. In systems of this type, the effector molecules directly bind to the target RNA, modulating its structure and affecting the expression of downstream coding sequence(s). In the S-box transcription termination control system, which is used primarily in low G+C Gram positive bacteria, binding of S-adenosylmethionine (SAM) to the leader RNA stabilizes an anti-antiterminator element that prevents formation of the antiterminator element, promoting the formation of the helix of an intrinsic transcriptional terminator. In Smk box RNAs, recently discovered in lactic acid bacteria, binding of SAM causes an RNA structural rearrangement that sequesters the Shine-Dalgarno (SD) sequence, resulting in translational control of gene expression. Each set of SAM-binding riboswitch RNAs exhibits a complex set of conserved sequence and structural elements in the 5’ region of the RNA that are essential for SAM-dependent regulation, but the two classes of RNAs differ markedly in structure. SAM recognition in both of these systems has been shown to be highly specific, but the molecular basis for binding specificity is not yet understood. This study was designed to further characterize the SAM binding properties of the S-box RNA, and to compare the binding properties of different SAM-binding RNAs in vivo and in vitro.
Keywords: S-adenosylmethionine, riboswitch, transcription termination
Abstract:
Modular RNA motifs that mediate tertiary interactions have been used to engineer artificial self-assembling RNA molecules to form nano- and meso-scopic structures, including closed cooperative oligomeric complexes, long, straight fibers, and two-dimensional arrays. Individual RNA tertiary interactions, such as the ubiquitous hairpin loop/receptor motifs, are relatively weak and readily reversible – with generally fast on- and off-rates – and are therefore promising components for designing RNA molecular machines. To achieve intermolecular self-assembly at sub-micromolar concentrations, loop/receptor interactions must be used in pairs. Each molecule must present two loop or receptor motifs, properly oriented for interaction with the cognate motifs in the partner molecule. Using two different binding loops, GAAA (L1) and GGAA (L2) and their cognate receptors, R1 and R2, only two unique, non-self-associating, helical inter-molecular interfaces can be engineered for directional assembly. To be correctly oriented for interaction the motifs must be separated by an integral number of helical turns. Strongest binding occurs with loops and receptors separated by one helical turn (~11 bp). One way to increase the combinatorial possibilities for RNA self-assembly using loop-receptor interactions is to find additional, loop-receptor interaction motifs that interact with the same geometry and that exhibit orthogonal specificity to existing motifs. Here we explore a second strategy, suggested by the identification in crystal structures of a new recurrent RNA motif, the C-loop, which locally increases the helical twist of an RNA helix in which it is embedded. With one embedded C-loop, a helix completes one turn in about nine base stacking layers, i.e. seven Watson-Crick basepairs and two base-triples belonging to the C-loop. We show that the same loop/receptor motif pairs could be used with C-loops to generate molecules that associate preferentially with other C-loop-containing molecules as compared to molecules having the same motifs but lacking C-loops.
Keywords: Tectons, C-loop, Assembly
Abstract:
The "Find RNA 3D" (FR3D) suite of programs exhaustively searches RNA atomic-resolution 3D structures for local and composite recurrent RNA 3D motifs, sets of RNA nucleotides with similar spatial arrangements. Local motifs comprise nucleotides in a single hairpin or internal loop. Composite motifs comprise nucleotides belonging to three or more different strand segments. FR3D uses a base-centered approach to efficiently, yet exhaustive search with geometric, symbolic, or mixed representations of RNA structure. Each base is represented by the position of its geometric center in 3D space and by the rotation matrix that describes its orientation with respect to a common frame. Base-pairing and -stacking interactions are calculated from the base geometries and represented using Leontis/Westhof bp classification. These data are stored for motif searches. For geometric searches, a user-supplied 3D query motif is used to find and score geometrically similar candidate motifs, without regard to the sequential position of their nucleotides in the RNA chain. A geometric discrepancy is defined to score and rank candidate motifs. Since it is impossible to explicitly compute the discrepancy for all conceivable candidate motifs in the RNA database, a screening algorithm was devised that is guaranteed to find all motifs having discrepancy below a user-specified cut-off. Candidate motifs identified geometrically may be screened symbolically for particular basepair or stacking arrangement, sequence continuity or identity constraints. Purely symbolic searches for motifs containing user-defined sequence, continuity and interaction constraints have also been implemented. Post-processing facilities allow the user to organize the candidate motifs into clusters and to display a multiple alignment of the nucleotide sequence corresponding to each candidate. The search algorithms have been optimized for speed to allow users to search the non-redundant RNA 3D structure database on a personal computer in a matter of minutes. FR3D is available at: http://rna.bgsu.edu/FR3D.
Keywords: Recurrent Motifs, Structural Bioinformatics
Abstract:
Recent evidence indicates translational control of inflammatory protein expression is important for resolution of chronic inflammation. In U937 cells, after 16 h of IFN-gamma (IFN) treatment, human ribosomal protein L13a is phosphorylated and released from the 60S ribosomal subunit. Phosphorylated L13a then interacts with 3 other proteins to form the IFN-Gamma Activated Inhibitor of Translation (GAIT) complex that binds a defined, 29-nt GAIT element in the 3’-UTR of ceruloplasmin (Cp) mRNA, and blocks its translation. L13a phosphorylation and GAIT complex activation occur almost simultaneously suggesting that L13a phosphorylation is the rate-limiting step in GAIT-mediated translational control. Kinase activity assays using lysates from IFN-treated U937 cells show delayed appearance of L13a-kinase activity after about 12-16 h, thereby explaining the delayed phosphorylation and recruitment of L13a to the GAIT complex, and consequent translational silencing of Cp mRNA. Immunoprecipitation of IFN-treated lysates with phosphoamino acid-specific antibodies followed by immunoblot using anti-L13a antibody, shows serine phosphorylation of L13a. Mass spectrometric analysis of recombinant, phosphorylated L13a from baculovirus-infected insect cells reveals Ser77 as a candidate phosphorylation site. This site was confirmed by the absence of phosphorylation of exogenous L13a in U937 cells transfected with L13a containing a Ser77-to-Ala mutation. Bioinformatic analysis suggests R-X-X-S77 as a possible kinase-recognition motif. In vitro phosphorylation of mutant peptides with cell lysates confirms the requirement for the Arg residue. Immunocomplex-kinase assay using IFN-treated 16 h cell lysate indicates death-associated protein kinase 3 (DAPK3) is responsible for L13a phosphorylation. SiRNA treatment confirmed both DAPK3, and its family member DAPK1, are involved in this phosphorylation cascade. Both kinases have putative GAIT element in their 3’-UTR and their expressions are regulated at the translational level after IFN-treatment. Therefore, we propose a negative feedback mechanism in which kinases responsible for L13a phosphorylation, and consequent translational silencing, are targets of the same regulatory pathway.
Keywords: GAIT, Ribosomal protein L13a, Phosphorylation
Abstract:
The catalytic domains of the aminoacyl tRNA synthetases (ARS) are of ancient origin and conserved across bacteria, archaea and eukaryotes. However, several ARSs have appended non-enzymatic domains, which may mediate non-canonical activities. The WHEP-TRS domain is a non-enzymatic domain found in five eukaryotic ARSs. In glutamyl-prolyl tRNA synthetase (EPRS) of animals, this domain exists in multiple repeats of variable number in the linker region between the two enzymatic domains. Results from our laboratory suggest that the WHEP-TRS domain of human EPRS is required for transcript-selective translational silencing and has both RNA-binding and protein-binding abilities. Investigating the origin and evolution of the WHEP domains can provide insights into the origin of EPRS and the evolution of its non-canonical activity. Previous analysis based on the presence of unlinked ERS and PRS in C. elegans had suggested that EPRS originated by a gene fusion event early in coelomate evolution, giving rise to EPRS with six WHEP repeats; subsequent deletions in vertebrates resulted in EPRS with three WHEP domains. We have cloned the EPRS WHEP domain from basal eukaryotic taxa (choanoflagellates and cnidarians) and performed protein phylogeny analysis using these sequences in combination with a large dataset of available WHEP sequences. Our results show for the first time that the bifunctional EPRS with WHEP domains originated by a gene fusion event before the origin of animals. Cloning and phylogenetic analysis of early chordate WHEP domains (cephalochordates and urochordates) suggest that the domain evolved by repeated duplications with divergence in multiple metazoan lineages. RNA-protein interaction studies using surface plasmon resonance suggest that the early WHEP domains had both mRNA and tRNA-binding functions. These data trace the origin of the only bifunctional ARS in eukaryotes. Independent duplication in multiple species and long-term persistence suggest that the WHEP repeats contribute to a unique mRNA-binding function of EPRS.
Keywords: tRNA synthetase, mRNA-binding domain, evolution
Abstract:
There are at least two routes for tRNA nuclear export in S. cerevisiae, but only the Los1p-dependent pathway is well described. Los1p, an unessential b-importin, binds tRNA in a RanGTP-dependent manner and exports tRNA out of the nucleus. We applied synthetic genetic array technology (Tong et al., 2001) to conduct a systematic, genome-scale search of unessential genes that function in Los1p-independent tRNA nuclear export. In addition to ARC1, a known interactor (Simos et al., 1996), >130 novel candidates were uncovered. As assessed by tetrad analysis and spot assay, 8 of 97 candidates have bonafide synthetic interactions. Three verified interactors, pho88-delta, gtr1-delta, and gtr2-delta, which cause tRNA nuclear accumulation, implicate inorganic phosphate (Pi) up-take in tRNA nucleus-cytosol distribution.
Synthetic sick or lethal interactions were expected to occur between los1-delta and mutants of the Los1-independent tRNA nuclear export pathway. However, pho88-delta cells accumulated cytoplasmic tRNA in their nuclei, which indicates that the synthetic interaction is due to induction of retrograde nuclear import of cytoplasmic tRNA and/or impaired re-export of tRNA to the cytosol.
Phosphate-uptake defects were phenocopied by growing wild-type cells in medium lacking Pi (SC -Pi) to take advantage of controlled starvation. Wild-type cells grown at 23 degrees C, in SC -Pi for 1 to 2 hours accumulated cytoplasmic tRNA within nuclei. This accumulation also occurred through induction of retrograde nuclear import of cytoplasmic tRNA and/or impaired re-export of tRNA to the cytosol. Whereas amino acid starvation has been shown to cause tRNA nuclear accumulation (Shaheen and Hopper, 2005), this is the first connection between Pi availability and tRNA nuclear-cytosol distribution.
Keywords: tRNA, transport
Abstract:
Arabidopsis possesses all genes corresponding to the mammalian polyadenylation factors. Of these, the gene encoding Arabidopsis CPSF 30 is complex one, encoding small (app. 28kD) and larger polypeptide (app. ~68 kD). The smaller form corresponds to Arabidopsis ortholog of eukaryotic CPSF30. CPSF30 and its yeast counterpart, Yth1p, are CCCH-type zinc finger proteins that have been shown to possess RNA-binding and nucleolytic activities. Homozygous mutants carrying a T-DNA insertion in the first exon of the Arabidopsis gene encoding CPSF30 are viable (Delaney et al 2006). However, while loss of expression of this gene is not lethal, the mutants show a stunted and auxin-tolerant phenotype. The bulk of poly(A) in the mutant is indistinguishable from that in the wild-type. These observations indicate that the Arabidopsis protein, which is encoded by a single-copy gene, is not absolutely essential for polyadenylation. Further studies have revealed that the purified Arabidopsis protein, like its Drosophila counterpart, possesses a novel endonuclease activity. This protein can cleave RNA in the absence of divalent metal cations indicating the lack of such requirement. However, the nuclease activity is significantly inhibited in the presence of zinc. The nuclease activity is interesting, as it is suggestive of a direct role of this protein in the 3'-end cleavage reaction. However, the inhibitory effects of zinc suggest that the "normal", intact protein may not be an active endonuclease. Current studies are focused on resolving these contrasting properties (RNA-binding vs. endonucleoytic) and better understanding the role of this protein in gene expression in plants.
References:
1. Delaney KJ, Xu R, Zhang J, Li QQ, Yun KY, Falcone DL, Hunt AG. Calmodulin interacts with and regulates the RNA-binding activity of an Arabidopsis polyadenylation factor subunit. Plant Physiol. 2006 Apr; 140(4):1507-21.
Keywords: Polyadenylation, CPSF30, Endonuclease
Abstract:
PMR1 is an mRNA endonuclease that catalyzes the selective decay of a discrete subset of mRNAs. In contrast to the generalized pathway of mRNA decay, which acts on non-translating mRNA, PMR1 acts by forming a complex with its translating substrate mRNA, where it initiates decay by endonuclease cleavage within the body of the mRNA. We previously showed that PMR1 is phosphorylated at Y650, and tyrosine phosphorylation at this site is required for targeting to polysomes and mRNA decay. Our current work identifies c-Src as the responsible kinase. Most tyrosine kinases form a complex with their substrate, and in this context can phosphorylate both the target protein and the kinase itself. In vitro kinase assays of PMR1-TAP complexes recovered from transfected cells identified 2 tyrosine-phosphorylated proteins, one of which disappears when recovery was performed with the Y650F mutant form of the protein. The remaining ~60 kDa protein was similar in size to c-Src, and its activity was blocked by the Src inhibitor PP2. Reciprocal co-ip experiments confirmed Src and PMR1 are recovered in the same complex, and more Src is recovered with the Y650F mutant than wild-type protein. In addition recombinant Src phosphorylates both PMR1 and a peptide with the tyrosine phosphorylation site in vitro. PMR1 does not target to polysomes in Src-deficient cells or cells expressing either kinase-inactive Src or a dominant negative form of Src. However, virtually all of the PMR1 targets to polysomes in cells expressing constitutively active Src. Similarly, Src activates PMR1-mediated mRNA decay whereas dominant-negative Src or catalytically-inactive Src do not. These data provide the first link between mRNA decay and signal transduction by tyrosine kinases.
Keywords: polysomal ribonuclease 1, c-Src kinase, mRNA decay
Abstract:
HDM2/p53 interaction controls p53 levels in cells, which is important for cell cycle progression and tumor development. Previous work in our lab has identified unique alternatively spliced form of MDM2 and MDM4, in response to UV, and cis-platinum but not to equitoxic levels of gamma radiation. The alternatively spliced variant of MDM2 can interfere with p53/MDM2 interaction by down-regulating MDM2 and in turn can activate p53. We have shown that stress induced alternative splicing of MDM2 and MDM4 is conserved between humans and mice. We also reported that formation of alternative splicing of MDM2 and MDM4 is through a specific pathway independent of p53, ARF, MDM2 over-expression and ATM/ATR kinases. To investigate the pathway, which induces alternative splicing we constructed human MDM2 mini-genes using the minimal regions of conserved sequences between MDM2 and MDM4 and their corresponding human homologs. These constructs have the ability to alternatively splice in vivo upon UV exposure. We also performed in vitro splicing of these 32P-labeled mini-gene transcripts using HeLa nuclear extract. In vitro splicing also results in the expected spliced products including the UV-induced alternatively spliced isoform. Using this assay we can map the minimal cis-elements and trans factors needed for alternative splicing of MDM2. Posttranslational modifications and changes in sub cellular localization of RNA processing proteins have been reported after cytotoxic stress. These changes can have global impact on RNA processing and may also regulate alternative splicing of MDM2. This study places alternative splicing of p53 modulators in the damage response pathway and introduces a new role for splicing regulators in the DNA damage response.
Keywords: alternative splicing, MDM2
Abstract:
Aminoacyl-tRNA synthetases (aaRSs) are responsible for attaching amino acids to their cognate tRNAs during protein synthesis. In eukaryotes, aaRSs are commonly found in multi-enzyme complexes, although the role of these complexes remains largely unknown. Associations between aaRSs have also been reported in archaea, including a complex between prolyl- and leucyl-tRNA synthetases (ProRS, LeuRS) in Methanothermobacter thermautotrophicus. Yeast two-hybrid screens suggested that lysyl-tRNA synthetase (LysRS) also associates with LeuRS in M. thermautotrophicus. Co-purification experiments confirmed that LeuRS, LysRS and ProRS associate in cell-free extracts. LeuRS bound LysRS and ProRS with comparable KDs of about 0.3 - 0.9 uM, further supporting the formation of a stable multi-synthetase complex. The steady-state kinetics of aminoacylation by LysRS indicated that LeuRS specifically reduces the Km for tRNALys over 3-fold, with no additional change seen upon addition of ProRS. No significant changes in the kinetics of aminoacylation by LeuRS or ProRS were observed upon addition of LysRS. These findings, together with earlier data, indicate the existence of a functional complex of three aminoacyl-tRNA synthetases in archaea in which LeuRS improves the catalytic efficiency of tRNA aminoacylation by both LysRS and ProRS.
Keywords: aminoacyl-tRNA synthetase
Abstract:
Protein synthesis requires the pairing of amino acids with tRNAs catalyzed by the aminoacyl-tRNA synthetases. The synthetases are highly specific, but errors in amino acid selection are occasionally made opening the door to inaccurate translation of the genetic code. The fidelity of protein synthesis is maintained by the editing activities of synthetases, which remove noncognate amino acids from tRNA before they can be delivered to the ribosome. While editing has been described in numerous synthetases, the reaction mechanism is unknown. To define the mechanism of editing, phenylalanyl-tRNA synthetase was used to investigate different models for hydrolysis of the noncognate product Tyr-tRNAPhe. Deprotonation of a water molecule by the highly conserved residue beta-His265, as proposed for threonyl-tRNA synthetase, was excluded since replacement of this and neighboring residues had little effect on editing activity. Model building suggested that instead of directly catalyzing hydrolysis, the role of the editing site is to discriminate and properly position non-cognate substrate for nucleophilic attack by water. In agreement with this model, replacement of certain editing site residues abolished substrate specificity but only reduced the catalytic efficiency of hydrolysis 2- to 10- fold. In contrast removal of the 3'-OH group of tRNAPhe severely impaired editing and revealed an essential function for this group in hydrolysis. The phenylalanyl-tRNA synthetase editing mechanism explains the discrepancy between the leucyl- and threonyl-tRNA synthetase models and provides a paradigm for synthetase editing.
References:
Jiqiang Ling, Hervé Roy and Michael Ibba. Mechanism of tRNA-dependent editing in translational quality control. Submitted.
Keywords: PheRS, editing mechanism, quality control
Abstract:
Eukaryotic cells are divided into multiple membrane-bound compartments, including the mitochondrion - the energy factory of aerobic organisms. Mitochondria possess their own genome that can vary in size, DNA content and structural arrangement amongst eukaryotes. Due to a dearth of mitochondrial genes the majority of proteins, needed for organellar function, are encoded in the nucleus and subsequently imported into the mitochondria. Likewise, in many organisms, the mitochondrial genome has an incomplete set of tRNA genes needed for translation. In these cases, tRNAs are also imported from the cytosol as an essential step for mitochondrial biogenesis. Here we show the first example of natively imported tRNAs into rat and human mitochondria. We found that two out of three nucleus-encoded tRNAGln isoacceptors localize to the mitochondria in vivo as confirmed by sequencing. Furthermore, we demonstrate that, like in yeast, in vitro import into isolated mammalian mitochondria occurs in an efficient and specific manner in the absence of added cytosolic factors. We conclude that tRNA import into eukaryotic mitochondria is more widespread than previously thought and it involves pathways that are mechanistically conserved from yeast to humans. Our findings also have direct implications for the study of a number of mitochondrial tRNA mutations that are causally linked to human disease.
Keywords: tRNA, Import, Mitochondria
Abstract:
The ribonucleoprotein enzyme RNase P processes all pre-tRNAs, yet some substrates apparently lack consensus elements for recognition. Here, we compare binding affinities and cleavage rates of Escherichia coli pre-tRNAs that exhibit the largest variation from consensus recognition sequences. These results reveal that the affinities of both consensus and non-consensus substrates for the RNase P holoenzyme are essentially uniform. Comparative analyses of pre-tRNA and tRNA binding to the RNase P holoenzyme and P RNA alone reveal differential contributions of the protein subunit to 5¡¯ leader and tRNA affinity. Additionally, these studies reveal that uniform binding results from variations in the energetic contribution of the 5¡¯ leader which serve to compensate for weaker tRNA interactions. Furthermore, kinetic analyses reveal uniformity in the rates of substrate cleavage that result from dramatic (>900-fold) contributions of the protein subunit to catalysis for some non-consensus pre-tRNAs. Together these data suggest that an important biological function of RNase P protein is to offset differences in pre-tRNA structure such that binding and catalysis are uniform.
Keywords: RNase P, C5, RNA
Abstract:
A widely held view is that directional movement of tRNA in the ribosome is determined by an intrinsic mechanism and driven thermodynamically by transpeptidation. Here, we show that, in certain ribosomal complexes, the pretranslocation (PRE) state is favored over the posttranslocation (POST) state at equilibrium. Spontaneous and efficient conversion from the POST to PRE state is observed when EF-G is depleted from ribosomes in the POST state or when tRNA is added to the E site of ribosomes containing P-site tRNA. In the latter assay, the rate of tRNA movement is increased by streptomycin and neomycin, decreased by tetracycline, and not appreciable affected by the acylation state of the tRNA. In one case, we provide evidence that complex conversion occurs predominantly by reverse translocation (i.e., direct movement of the tRNAs from the E and P sites to the P and A sites, respectively). These findings have implications for the energetics of translocation.
Keywords: Ribosome, Translocation, Antibiotics
Abstract:
RNA chaperone activities are essential for virtually every process containing RNA to facilitate RNA conformational changes and to assist the assembly and disassembly of RNA-RNA and RNA-protein interactions. Despite the importance of RNA chaperones, there are few mechanistic studies that further our understanding of their mode of action. To overcome these difficulties we developed a mechanistic framework to study the formation of two short hybrids between the U3 snoRNA (the RNA part of the small subunit processome) and complementary sites of the pre-rRNA. In a previous study we demonstrated that two proteins, Imp3p and Imp4p, chaperone formation of both interactions. Formation of these two hybrids is essential to initiate small ribosomal subunit biogenesis. Moreover, these hybrids represent prototypes of interactions that require a chaperone.
To further our understanding of RNA chaperone action we developed a kinetic and thermodynamic framework that enables to discriminate between common chaperone mechanisms. First, we developed FRET based assays to show that Imp3p and Imp4p assemble into a ternary complex in an ordered and RNA-dependent manner, consistent with yeast two hybrid data. Second, we measured hybridization rates and duplex affinities for both hybrids. These assays revealed that the ternary complex uses product capture mechanism to facilitate hybridization at one site, thus increasing the stability of an otherwise unstable duplex by a thousand fold. We showed that hybridization at the other site is a two-step process: first, the ternary complex unfolds the U3 stem presumably to expose the relevant bases for hybridization and second, the ternary complex increases the hybridization rate by lowering the free energy of the transition state.
Hybridization rates measured are in agreement with the timeframe these interactions are expected to form in vivo. We propose that our mechanistic framework can be used to decipher the mechanism of other RNA chaperones.
References:
Gérczei, T. and Correll, C.C. (2004) Imp3p and Imp4p mediate an essential U3-pre-rRNA hybridization, presumably to recruit the small subunit processome to the pre-rRNA. Proc Natl Acad Sci U S A 101 (43), 15301-15306
Keywords: chpaerone, folding, ribosome biogenesis
Abstract:
Assembly of functional 30 S subunits is dependent on ribosomal protein (rprotein) - ribosomal RNA (rRNA) interactions that facilitate and stabilize folding of 16 S rRNA into the active conformation. Binding sites for several rproteins are located in the central domain of 16 S rRNA. S15, a central domain primary binding protein, has been shown to trigger a conformational change in the rRNA. This conformational change allows other central domain proteins - S6, S18, S11 and, S21 - to bind to the region causing a cascade of changes resulting in the functional structure of the central domain.
Biochemical and structural studies have revealed two regions that are minimally needed for binding S15 in vitro. One of the regions is the junction of helices 20, 21, and 22. The junction includes nucleotides 652-654 and 752-754 plus two or three base pairs in each helix for maintaining the junction structure. All junction nucleotides except 653 are highly conserved in bacteria implying these nucleotides are functionally important. Mutagenesis studies suggest that the junction plays a role in S15 recognition of 16 S rRNA. In particular, 752 mutants have decreased affinity for S15.
The interaction of the junction loop (652-654 and 752-754) with S15 does not appear to be base-specific; instead, S15 binds to the backbone of nucleotides 752-754. Interactions between nucleotides 652-654 and 752-754 create a structure that places the backbone in the proper position to bind S15. Conservation of junction nucleotides possibly occurs because single nucleotide changes would disrupt S15 binding. To identify alternative functional junction loop sequences and the common structural motif, a saturation mutagenesis experiment of nucleotides 652-654 and 752-754 was performed.
Nucleotides 652-654 and 752-754 were randomly mutated and functional sequences were selected. Sixty-one unique mutants were isolated in the selection, sequenced, and assayed for function. No nucleotide was excluded at any position. The wild-type nucleotide appears to be preferred at each position except at 653. 653 had a non-random distribution of nucleotides with adenosine being preferred. Single-site mutagenesis at 653 showed no preference for nucleotide identity.
Keywords: ribosomal protein S15, 30 S subunit central domain
Abstract:
Ribosomal RNAs are the functionally important components of ribosomes. Helix 23b is located in the small-subunit rRNA and plays an important role in interactions of tRNA with the P-site and the E-site, mRNA binding, IF3 binding, and formation of the initiation complex. In addition, helix 23 is involved in intramolecular interactions with helix 24 (the 790 loop) and intermolecular interactions with ribosomal proteins and domain IV of 23S rRNA of large ribosomal subunit. The hairpin loop nucleotides of helix 23 are highly phylogenetically conserved. The nucleotide sequence of helix 23 internal loop (nucleotides 684 to 687 and 700 to 706 of Escherichia coli 16S rRNA), however, differ significantly between animals and bacteria.
The aim of this project is to identify the functionally important sequence and structural motifs within the internal loop of helix 23. This will be accomplished by integrating in vivo genetic studies with structural studies using NMR.
For genetic studies, eleven nucleotides of helix 23b internal loop were randomly mutated using PCR. The PCR products were cloned into cloning vector pwk122. A total of 3 x 107 clones were obtained to ensure the representation of mutant bank with > 99.9% confidence. The Helix 23b internal loop mutant bank was moved into expression vector pRNA228. In this vector, the chloramphenicol acetyltransferase (CAT) and the green fluorescent protein (GFP) mRNAs are translated only by plasmid derived ribosomes. CAT protein renders selectivity of mutants and GFP allows rapid and accurate determination of ribosome function by directly measuring fluorescence in whole cells. Functional mutants were selected , sequenced and assayed for GFP% function. A total of 115 unique functional mutants were isolated in the range of 13%-139% of wild type GFP function. The number of mutations per survivor ranged from two to eight nucleotides.
Keywords: Helix23b 16S rRNA, GFP function
Abstract:
One region of the ribosome of high interest is helix 69 of 23S rRNA. Helix 69 of Escherichia coli (E. coli) 23S rRNA has three Ø residues at positions 1911, 1915 and 1917. The Ø residue at position 1915 is methylated. This helix resides at the interface of the large and small subunits of the ribosome. A large contact area of the ribosomal inter-subunit bridge B2a includes helix 69, for which the crystal structure shows slightly different conformations of the loop region.1 It is proposed that helix 69 is important for ribosomal release. Furthermore, it has been suggested that the binding of ribosomal recycling factor changes the conformation of the helix 69. Our work involves understanding the dynamic nature of helix 69 and contribution of pH to its structure. It is hypothesized that changes in pH may result in conformational changes of the helix 69 structure. Biophysical methods including circular dichroism spectroscopy, thermal melting analysis and NMR spectroscopy were used to explore the pH-dependent changes in helix 69 structure, and to determine if the pH effects are localized to specific microenvironments within helix 69 involving the modified nucleotides. Comparisons were made between the wild-type helix 69 and the unmodified helix 69 construct, in which pseudouridines and 3-methylpseudouridine were replaced by uridines. These comparisons emphasize the importance of the pseudouridine modification in structure and dynamics of helix 69.
References:
1. Schuwrith, B.S., Borovinskya, M.A., Hau, C.W., Zhang, W., Vila-Sanjurjo, A., Holton, J.M., and Cate, J.H.D. 2005. Structures of the bacterial ribosome at 3.5 Å resolution. Science 310: 827-834.
Keywords: Helix 69, Pseudouridine
Abstract:
An understanding of structural dynamics is essential to the full characterization of any biomolecule, but is especially relevant with respect to RNA for which dynamics is used in myriad ways to achieve functional complexity that would otherwise be inaccessible based on a rigid framework composed of only four chemically similar nucleotides. Due to experimental difficulties in resolving the manifold of motional modes that pervade RNA structures, their dynamical properties remain poorly understood. Solution NMR is one of the most powerful tools for the characterization of structural dynamics, as it provides atomic level detail on a variety of timescales.
Spin relaxation measurements can provide information on motions occurring at ps to ns timescales. This includes bond librations, local base and sugar fluctuations, and global movements of helical domains. Traditionally, spin relaxation data is interpreted using to the Model Free formalism. At the crux of this formalism is the assumption that internal motions are not correlated to overall rotational diffusion. Owing to their extended architecture and prevalence of motions occurring at timescales approaching overall rotational diffusion, this approximation often breaks down for RNA often rendering interpretation of relaxation data intractable. It was recently shown that a domain elongation strategy can be used to eliminate couplings between internal motions and overall diffusion (Zhang et al., Science (2006) 311, 653-656). Using N-15 relaxation data measured in Watson-Crick residues, diffusion limited inter-domain motions were observed in the transactivation response element (TAR) from HIV-1 that evade detection in non-elongated constructs.
Here we have used domain-elongation in conjunction with C-13 relaxation measurements in both sugar and base moieties to characterize motional modes inaccessible by N-15 relaxation data. Model free analysis of these results using spectral density functions that account for asymmetric CSAs and anisotropic tumbling (i) reaffirm the existence of domain motions occurring at nanosecond diffusion limited timescales and (ii) reveal extremely large variations in the amplitude of local motions with unprecedented degrees of mobility observed for bulge residues that act as the hinge site for domain motions. The site-specific dynamical parameters are quantitatively interpreted in terms of TAR’s ability to adopt different conformations following adaptive recognition.
References:
Zhang et al., Science (2006) 311, 653-656
Keywords: NMR Relaxation, Dynamics, ModelFree
Abstract:
SL-1 is a highly conserved stem-loop RNA element that plays an essential role in the dimerization and non-covalent attachment of two copies of the HIV viral genome which must be specifically packaged during viral assembly. The function of SL-1 is believed to involve a dynamical structural transition between metastable kissing and thermodynamically stable duplex forms of the SL1 dimer. This structural isomerization can occur spontaneously, has been shown to be dependent on the presence of a highly conserved SL-1 internal bulge and Mg2+ divalent cations, and is believed to be catalyzed in vivo by the nucleocapsid protein. Using NMR, including residual dipolar couplings and motionally decoupled relaxation data, we characterized the structural plasticity of the bulge-containing SL-1 monomer and its dependence on Mg2+ binding. We observe a combination of local and global nanosecond and micro-to-millsecond motions in free SL-1 that are arrested upon Mg2+ binding to the bulge. Our results rationalize in vitro studies on spontaneous structural isomerization including dependence on the presence/absence of the bulge and Mg2+ and suggest that the role of NcP in isomerization may involve the ejection of stabilizing Mg2+ ions that are bound at the guanine rich internal bulge.
Keywords: RNA dynamics, NMR, Mg effect
Abstract:
Comparing to protein, RNA crystallization is a much more challenging task. Among the extra features presented in RNA, the most prominent is the negatively charged backbone that adopt intricate scaffold and might make crystal packing more difficult. We are trying to tackle this problem by co-crystallize the RNA with its Antigen Binding Fragment (FAB). Using a reduced codon antibody FAB library displayed on the M13 phage, we have selected FABs that bind to Delta C209 P4-P6 domain of Tetrahymena Group I intron. Two FABs, FAB2 and FAB5, bind to the Delta C209 with affinities of 50 nM and 30 nM, respectively. These FABs are highly specific and do not bind to BP, a Delta C209 mutant, in which tertiary RNA folding has been disrupted. Furthermore, the binding between these FABs and Delta C209 is diminished when [Mg2+] was reduced to zero, showing that these FABs bind to the tertiary structure of Delta C209. FAB2 was cocrystallized with Delta C209 and the structure was solved at 1.95 Å resolution. The Fe-EDTA footprinting assay and the crystal structure reveal that FAB2 does not alter the overall folding of Delta C209 either in solution or in crystal. The crystal structure also shows that, with direct and water-mediated hydrogen bonding network, FAB2 helps Delta C209 achieve its native folding with fewer innersphere coordinated magnesium ions. The protein participated crystal contacts account for 61% of the buried surface area and we expect this method will facilitate the crystallization of RNA by providing crystal contacts and lending structure stability.
Keywords: Chaperone-Assisted RNA Crystallization, RNA Antibody, Phage Display
Abstract:
Metagenomics has been employed to systematically sequence, classify, analyze, and manipulate the entire genetic material isolated from environmental samples. Finding genes within metagenomic sequences remains a formidable challenge and noncoding RNA genes other than those encoding rRNA and tRNA are not well annotated in metagenomic projects. In this work, we identify, validate, and analyze the genes coding for RNase P RNA (P RNA) from all published metagenomic projects. P RNA is the RNA subunit of a ubiquitous endoribonuclease RNase P that consists of one RNA subunit and one or more protein subunits. The bacterial P RNAs are classified into two types, Type A and Type B, based on the constituents of the structure involved in precursor tRNA binding. Bacterial and some archaeal P RNAs are catalytically active without protein subunits, capable of cleaving precursor tRNA transcripts to produce their mature 5΄-termini. We have found 328 distinctive P RNAs (320 bacterial and 8 archaeal) from all published metagenomics sequences, which led us to expand by 60% the total number of this catalytic RNA from prokaryotes. Surprisingly, all newly-identified P RNAs from metagenomics sequences are of Type A. One of the distinctive features of some new P RNAs is that the P2 stem has kinked nucleotides in its 5’ strand. We find that the single nucleotide J2/3 joint region linking the P2 and P3 stem that was used to distinguish a bacterial P RNA from an archaeal one is no longer applicable, i.e. some archaeal P RNAs have only one nucleotide in the J2/3 joint. We also discuss the phylogenetic analysis based on covariance model of P RNA that offers a few advantages over the one based on 16S rRNA.
Keywords: rnase p rna, metagenomics
Abstract:
Many viruses of plants use RNA as a genetic material, allowing for studies on template function of RNA. The viral RNA contains cis-acting regulatory sequences to control RNA synthesis. RNA replication in the virus-infected cells is performed via assembly of a specialized viral RNA-protein complex, named viral replicase complex. To study the assembly of the functional replicase complex (RC) for Tomato bushy stunt virus (TBSV), a small model plant virus, we expressed His6-tagged p33 and p92pol replication proteins and a TBSV replicon RNA in yeast, a model host. Affinity-based purification of the replicase complex from solubilized yeast membrane fractions was used to obtain functional replicase preparations. Based on in vitro replicase assays with added RNA templates, we found that three short cis-acting elements had to be present in the expressed viral RNA for the assembly of the viral RC. These were the p33 recognition element (p33RE), a replication promoter (gPR) and a replication silencer element (RSE). Detailed analysis of the role of these sequences revealed that long-range interaction might be important for the assembly of the RC. Building on our recent data, the most current model on the assembly of the tombusvirus RC will be discussed.
References:
1. Pogany, J., Fabian, M., White, K.A., and Nagy, P. D. 2003. A replication silencer element in an RNA virus. EMBO Journal 22: 5602-5611.
2. Panaviene, Z., Panavas, T., and P. D Nagy. 2005. Role of an internal and two 3\'-termi
Keywords: viral RNA, RNA-protein interaction, secondary structure
