RNA's modified nucleosides are essential to protein synthesis. However, too little is known of the biochemical and structural contributions of the 100 natural, post-transcriptional modifications to relate structure to function. Fundamental understandings of modified nucleoside functions have shown promise in identifying novel therapeutic targets in infectious disease, and in optimizing ribozyme, antisense and aptamer activities. Thus, this project's long-term objective is to understand how the biochemical and structural contributions of modified nucleosides impact protein recognition of RNA and its function in translation. tRNA, composed of physically and functionally separable domains that are easily investigated for modification-dependent structure/function relationships, is an excellent model for this study. We hypothesize that anticodon modified nucleosides provide a common architecture for stably recognizing mRNA coding triplets on the ribosome, while at the same time providing hydrophobic, hydrophilic and/or electrostatic properties as identity elements for aminoacyl-tRNA synthetase (aaRS) recognition. Using methods developed almost exclusively by us for the automated chemical synthesis of RNA with site- selectively placed modified and stable isotope labeled nucleosides, we have found three functionally important modified nucleosides that modulate anticodon loop architecture. To determine the structure/function relationships of other anticodon domain modifications, we will: 1) Identify and characterize the modified nucleoside-dependent ribosomal binding of tRNA anticodon domains that do not bind in their unmodified sequences; 2) Determine and characterize the properties of modified anticodon domains that are selectively recognized by peptides as mimics of aaRS; and 3) Characterize the structural properties contributed by modified nucleosides to the ribosomal binding and aaRS recognition of anticodons. Our approach will utilize a unique combination of chemical synthesis, biochemical assays of function and thermal denaturation, calorimetry, fluorescence, CD and NMR spectroscopy for determining stability and structure to distinguish those modifications required for ribosome binding and aminoacylation and their critical physiochemical contributions.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM023037-20
Application #
6621999
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Flicker, Paula F
Project Start
1988-01-01
Project End
2006-01-31
Budget Start
2003-02-01
Budget End
2004-01-31
Support Year
20
Fiscal Year
2003
Total Cost
$241,725
Indirect Cost
Name
North Carolina State University Raleigh
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
042092122
City
Raleigh
State
NC
Country
United States
Zip Code
27695
Xiao, Xingqing; Agris, Paul F; Hall, Carol K (2015) Designing peptide sequences in flexible chain conformations to bind RNA: a search algorithm combining Monte Carlo, self-consistent mean field and concerted rotation techniques. J Chem Theory Comput 11:740-52
Harris, Kimberly A; Bobay, Benjamin G; Sarachan, Kathryn L et al. (2015) NMR-based Structural Analysis of Threonylcarbamoyl-AMP Synthase and Its Substrate Interactions. J Biol Chem 290:20032-43
Halvorsen, Ken; Agris, Paul F (2014) Cross-platform comparison of nucleic acid hybridization: toward quantitative reference standards. Anal Biochem 465:127-33
Spears, Jessica L; Xiao, Xingqing; Hall, Carol K et al. (2014) Amino acid signature enables proteins to recognize modified tRNA. Biochemistry 53:1125-33
Rodriguez-Hernandez, Annia; Spears, Jessica L; Gaston, Kirk W et al. (2013) Structural and mechanistic basis for enhanced translational efficiency by 2-thiouridine at the tRNA anticodon wobble position. J Mol Biol 425:3888-906
Harris, Kimberly A; Shekhtman, Alexander; Agris, Paul F (2013) Specific RNA-protein interactions detected with saturation transfer difference NMR. RNA Biol 10:1307-11
Vendeix, Franck A P; Murphy 4th, Frank V; Cantara, William A et al. (2012) Human tRNA(Lys3)(UUU) is pre-structured by natural modifications for cognate and wobble codon binding through keto-enol tautomerism. J Mol Biol 416:467-85
Graham, William D; Barley-Maloney, Lise; Stark, Caren J et al. (2011) Functional recognition of the modified human tRNALys3(UUU) anticodon domain by HIV's nucleocapsid protein and a peptide mimic. J Mol Biol 410:698-715
Bilbille, Yann; Gustilo, Estella M; Harris, Kimberly A et al. (2011) The human mitochondrial tRNAMet: structure/function relationship of a unique modification in the decoding of unconventional codons. J Mol Biol 406:257-74
Harris, Kimberly A; Jones, Victoria; Bilbille, Yann et al. (2011) YrdC exhibits properties expected of a subunit for a tRNA threonylcarbamoyl transferase. RNA 17:1678-87

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