The objective of this project is to study the biochemical properties of tRNA molecules prepared by in vitro transcription that have defined structural modifications at various positions in the polynucleotide chain. These experiments will permit a better understanding of how tRNA is recognized by the enzymes that interact with it. It is possible that certain mutant tRNAs will block an enzyme reaction pathway at a new step, revealing more about the mechanism of the enzyme. Finally, since the structure of the mutant RNAs will be carefully studied, more will be learned about the folding of RNAs into a tRNA-like structure. Mutant E. coli tRNAs will be assayed on E. coli ribosomes using several equilibrium and kinetic assays for binding, dipeptide formation and proofreading steps. tRNA nucleotides involved in specific ribosome tRNA contacts will be identified. The nucleotides important for the recognition of yeast and E. coli tRNA Phe by their cognate synthetases will be compared. The available co-crystal structure of the E. coli glutamine tRNA-enzyme complex will be used to design experiments to understand a conformational isomerization that appears to occur in the reaction pathway. RNAs which are expected to fold like tRNA but have sequences very different from tRNA will be designed and studied. The structure and activity of several isolated tRNA domains will be studied.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Biochemistry Study Section (BIO)
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University of Colorado at Boulder
Schools of Arts and Sciences
United States
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Shepotinovskaya, Irina; Uhlenbeck, Olke C (2013) tRNA residues evolved to promote translational accuracy. RNA 19:510-6
Chapman, Stephen J; Schrader, Jared M; Uhlenbeck, Olke C (2012) Histidine 66 in Escherichia coli elongation factor tu selectively stabilizes aminoacyl-tRNAs. J Biol Chem 287:1229-34
Saks, Margaret E; Sanderson, Lee E; Choi, Daniel S et al. (2011) Functional consequences of T-stem mutations in E. coli tRNAThrUGU in vitro and in vivo. RNA 17:1038-47
Schrader, Jared M; Uhlenbeck, Olke C (2011) Is the sequence-specific binding of aminoacyl-tRNAs by EF-Tu universal among bacteria? Nucleic Acids Res 39:9746-58
Dale, Taraka; Fahlman, Richard P; Olejniczak, Mikolaj et al. (2009) Specificity of the ribosomal A site for aminoacyl-tRNAs. Nucleic Acids Res 37:1202-10
Schrader, Jared M; Chapman, Stephen J; Uhlenbeck, Olke C (2009) Understanding the sequence specificity of tRNA binding to elongation factor Tu using tRNA mutagenesis. J Mol Biol 386:1255-64
Ledoux, Sarah; Olejniczak, Miko?aj; Uhlenbeck, Olke C (2009) A sequence element that tunes Escherichia coli tRNA(Ala)(GGC) to ensure accurate decoding. Nat Struct Mol Biol 16:359-64
Ledoux, Sarah; Uhlenbeck, Olke C (2008) Different aa-tRNAs are selected uniformly on the ribosome. Mol Cell 31:114-23
Ledoux, Sarah; Uhlenbeck, Olke C (2008) [3'-32P]-labeling tRNA with nucleotidyltransferase for assaying aminoacylation and peptide bond formation. Methods 44:74-80
Sanderson, Lee E; Uhlenbeck, Olke C (2007) Directed mutagenesis identifies amino acid residues involved in elongation factor Tu binding to yeast Phe-tRNAPhe. J Mol Biol 368:119-30

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