During the reading of the genetic code, bacteria use more than 50 different aminoacyl-tRNAs as interchangeable substrates of the ribosome. The long-term goal of this project is to understand why each bacterial tRNA species has a unique, phylogenetically conserved set of nucleotides and post-transcriptional modifications. The project will test the hypothesis that most of this structural diversity has evolved to idiosyncratically "tune" each tRNA species such the ribosome can decode the different codons with similar rates and minimize misreading of incorrect codons. In other words, individual tRNAs are different so that they can be equivalent substrates of the ribosome and perform accurately in translation. The project will use a combination of biochemistry, biophysics, and molecular genetics to identify the tRNA sequences and post-transcriptional modifications that are responsible for tuning and then understand their mechanism. Several experiments will exploit our recent discovery that the three base pairs in T-stem of tRNA that ensure uniform binding to elongation factor Tu also affect the release of aminoacyl-tRNA from elongation factor Tu on the ribosome. Three different E. coli tRNAs have been chosen for a large scale mutagenic screen for sequence elements that tune tRNAs in decoding by the ribosome. Finally, five poorly studied modifications present in the body of tRNA will be examined for their role(s) in ribosomal decoding. Taken together, these experiments should for the first time provide a comprehensive understanding of how different tRNAs adapt to the ribosome.

Public Health Relevance

Since bacterial infections are an important human health problem, this basic research defining the fundamental properties of tRNA molecules may lead to more efficient control of bacterial growth. Many of the principles deduced for bacterial tRNAs are expected to be true for human tRNAs as well.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM037552-26
Application #
8447592
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Bender, Michael T
Project Start
1986-02-01
Project End
2014-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
26
Fiscal Year
2013
Total Cost
$381,346
Indirect Cost
$131,283
Name
Northwestern University at Chicago
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
160079455
City
Evanston
State
IL
Country
United States
Zip Code
60201
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
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, Mikolaj; 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
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
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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