The goal of this proposal is to understand the relationship between the structure of tRNA and its function in aminoacylation and protein synthesis. We have developed efficient enzymatic procedures to cleave tRNA in the anticodon loop, remove one or more nucleotides, insert other nucleotides and reseal the tRNA to produce a variant molecule with a defined chemical change in the anticodon. Similar procedures will be developed to alter other parts of the tRNA molecule. The substrate sepcificity of the enzymes used in this procedure are sufficiently broad that a wide variety of base and sugar modifications can be inserted into the polynucleotide chain. In addition, an in vitro transcription procedure will be developed to obtain unmodified but fully processed tRNAs from tRNA gene. Taken together, these methods give a high degree of synthetic control over a crucial molecule in the translation mechanism. The methods should be applicable to other RNA molecules. We will use this technology to prepare variant tRNAs to examine several aspects of tRNA function. First, we want to evaluate the relative role of different regions of tRNA in the specificity of its interaction with its cognate tRNA synthetase. The kinetics of aminoacylation of tRNAs with modifications in the anticodon, the D stem region and the acceptor stem region will be compared. Second, the codon-anticodon interaction of tRNAPhe will be examined on poly U programmed E. coli ribosomes. Steady state and single turnover kinetics of phenylalanine tRNAs modified in the anticodon loop will be determined.

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National Institute of General Medical Sciences (NIGMS)
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Biophysics and Biophysical Chemistry A Study Section (BBCA)
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University of Colorado at Boulder
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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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