Molecules of tRNA translate codons in mRNA into the amino acid sequences of protein. An essential feature of this process is the enzymatic aminoacylation of tRNA. Our long term interest is to determine which aspects of tRNA structure determine aminoacylation specificity. Our general approach is to use computer analysis of tRNA sequences to predict likely determinants, and to test those predictions with mutant tRNAs expressed in Escherichia coli. The aminoacylation specificity of tRNA is ascertained by determining the amino acid inserted in a protein corresponding to the codon read by the mutant tRNA. Two considerations have led us to adopt this approach. First, computer analysis of tRNA sequences reduces the very large number of possible specificity determinants to a testable number. Second, in vivo analysis of tRNA reflects the outcome of cognate and noncognate aminoacylating enzymes competing for the tRNA. The power of this approach has been demonstrated by our successes in elucidating the determinants for several tRNAs. Furthermore, because determinants of tRNA aminoacylation are often conserved, our structure-function analysis, which is most feasible in E. coli, should have broad applicability. During the proposed granting period, we will: 1. Define the aminoacylation determinants of several E. coli tRNAs using nonsense suppressor tRNAs. 2. Develop and implement a system to study missense suppressor tRNAs in E. coli. Having realized that a given tRNA often contains determinants in its anticodon and acceptor end, we will develop a missense tRNA system capable of analyzing determinants in both locations simultaneously; this is not possible with nonsense suppressor tRNAs. 3. Genetically characterize the mode of action of determinants distant from the site of catalysis in the acceptor end of tRNA. Since remote (less than 60 Angstroms) determinants affect catalysis, an allosteric change in tRNA and/or the enzyme structure is implied. We will use a genetic approach to investigate this structure-function relationship in tRNA by selecting secondary mutants that compensate for defects in remote determinants.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Physiological Chemistry Study Section (PC)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Wisconsin Madison
Schools of Earth Sciences/Natur
United States
Zip Code
McClain, William H (2006) Surprising contribution to aminoacylation and translation of non-Watson-Crick pairs in tRNA. Proc Natl Acad Sci U S A 103:4570-5
Lee, Dennis; McClain, William H (2004) Aptamer redesigned tRNA is nonfunctional and degraded in cells. RNA 10:7-11
McClain, William H; Gabriel, Kay; Lee, Dennis et al. (2004) Structure-function analysis of tRNA(Gln) in an Escherichia coli knockout strain. RNA 10:795-804
Choi, Hyunsic; Gabriel, Kay; Schneider, Jay et al. (2003) Recognition of acceptor-stem structure of tRNA(Asp) by Escherichia coli aspartyl-tRNA synthetase. RNA 9:386-93
Choi, H; Otten, S; McClain, W H (2002) Isolation of novel tRNA(Ala) mutants by library selection in a tRNA(Ala) knockout strain. Biochimie 84:705-11
Choi, Hyunsic; Otten, Sharee; Schneider, Jay et al. (2002) Genetic perturbations of RNA reveal structure-based recognition in protein-RNA interaction. J Mol Biol 324:573-6
McClain, W H; Gabriel, K (2001) Construction of an Escherichia coli knockout strain for functional analysis of tRNA(Asp). J Mol Biol 310:537-42
Gabriel, K; McClain, W H (2001) Plasmid systems to study RNA function in Escherichia coli. J Mol Biol 310:543-8
Moulinier, L; Eiler, S; Eriani, G et al. (2001) The structure of an AspRS-tRNA(Asp) complex reveals a tRNA-dependent control mechanism. EMBO J 20:5290-301
Chang, K Y; Varani, G; Bhattacharya, S et al. (1999) Correlation of deformability at a tRNA recognition site and aminoacylation specificity. Proc Natl Acad Sci U S A 96:11764-9

Showing the most recent 10 out of 29 publications