2013 Rustbelt RNA Meeting







Talk abstracts

Talk on Saturday 11:45-12:00pm submitted by Courtney Niland

Investigation of Shared Molecular Recognition of Pre-tRNAs By RNA and Protein Components of RNase P Using High-Throughput Sequencing Kinetics (HTS-Kin)

Courtney N. Niland (Department of Biochemistry, Case Western Reserve University), Ulf-Peter Guenter (Department of Biochemistry, Case Western Reserve University, Center for RNA Molecular Biology, Case Western Reserve University), Eckhard Jankowsky (Department of Biochemistry, Case Western Reserve University, Center for RNA Molecular Biology, Case Western Reserve University), Michael E. Harris (Department of Biochemistry, Case Western Reserve University)

Ribonucleoprotein enzymes, such as the ribosome, spliceosome, RNase P, RISC Complex, and snRNPs, are essential to life and must interact with a large number of alternative substrates to function. Ribonuclease P , RNase P recognizes all precursor tRNAs (pre-tRNAs) that differ in sequence and structure to perform its role in 5prime end maturation. Work from our lab and others reveals that both the protein and catalytic RNA subunits of RNase P possess sequence specificity in contacts to the 5prime leader of pre-tRNA. We are investigating how variation in the 5prime leader of pre-tRNAs affects binding by the holoenzyme and ribozyme and how these contacts influence one another. We developed a new method allowing us to quantify individual rate constants for a reaction containing a large number of alternative substrates. The power of this technique is the ability to analyze all possible sequences in areas of protein and RNA contact, providing information about thermodynamic coupling. By randomizing regions of the 5prime leader contacting the protein (nucleotides -3 to -8) or the protein and RNA (nucleotides -1 to -6), we can investigate each population for processing by the RNase P ribozyme and holoenzyme. Next-generation sequencing of the residual substrate population along the reactions allows us to calculate the rate constants of each substrate sequence. Comparison of ribozyme and holoenzyme reactions with the same substrate population shows changes in preferential sequence in regions of RNA and protein contact. This combined with the changing shape of the affinity distribution indicates that this crosstalk is occurring. This information will provide key insight into the molecular recognition of RNase P by investigating thermodynamic and kinetic coupling in the RNA-RNA and protein-RNA contacts present in RNase P substrate recognition.

U. Guenther, L.E. Yandek, C.N. Niland, F.E. Campbell, D. Anderson, V.E. Anderson, M.E. Harris, and E. Jankowsky. (2013) Hidden Specificity In An Appartently Non-Specific RNA-Binding Protein. Nature (in press).

L.E. Yandek, H.C. Lin, and M.E. Harris. (2013) Alternative Substrate Kinetics of Eschericia coli Ribonuclease P: Determination of Relative Rate Constants By Internal Competition. J. Biol. Chem. Mar 2013;288(12):8342-54

L. Sun, F.E. Campbell, L.E. Yandek, and M.E. Harris. (2010) Binding of C5 Protein to P RNA Enhances The Rate Constant For Catalysis For P RNA Processing of pre-tRNAs Lacking A Consensus G(+1)/C(+72) Pair. J. Mol. Biol. Feb 2010;395(5):1019-37

N.H. Zahler, E.L. Christian, and M.E. Harris. (2003) Recognition of the 5prime leader of pre-tRNA Substrates By The Active Site of Ribonuclease P. RNA. Jun 2003;9(6):734-45

Keywords: Ribonuclease P, Alternative Substrate Kinetics, RNA Processing