There is a wealth of information available on the chemical nature of modifications in tRNA, including their contributions to translational accuracy and efficiency. Despite this body of data, very little is known about the structural effects of modifications on tRNA and the thermodynamic contributions of modifications to RNA- RNA interactions outside the ribosome. Indeed, NMR studies have shown that unmodified anticodon arms can adopt spectrum of conformations. In Gram-positive bacteria, tRNA molecules not only shuttle amino acids to the ribosome for protein synthesis, but also are the sensors through which transcription of aminoacyl tRNA synthetase genes and genes involved in amino acid metabolism are regulated. In the mRNA of these genes, a leader region upstream from the translation start site binds tRNA in a codon specific fashion. This mRNA leader, known as the T-box, regulates expression of the gene through a transcriptional antitermination mechanism in a tRNA-dependent fashion. A bound tRNA that is uncharged prevents formation of a transcriptional terminator and allows read-through by RNA polymerase whereas a bound tRNA that is charged leads to transcription termination. The interaction between the tRNA anticodon and the leader RNA is critical to the function of this regulatory system and it is well established that modifications in the anticodon arm of tRNA modulate codon-anticodon interactions on the ribosome.
The aims of this proposal are: (1) to determine by NMR the conformational and dynamical effects of base modification on the anticodon arms of tRNA (2) to determine the energetics of anticodon-leader RNA association as a function of modification state using ITC (3) to determine by NMR the structures of anticodon arm-leader RNA complexes (4) to determine the antitermination efficiencies of variously modified tRNA molecules in vitro using a purified antitermination assay and in vivo using modification deficient bacterial strains and (5) to characterize the physiology of loss-of-modification mutants in a Gram-positive pathogen. These studies are designed to identify correlations between the physical effects of tRNA base modification on RNA-RNA recognition and on the biological activity of tRNA molecules. A thorough knowledge of pathogen-specific modification may also define new targets for antimicrobial strategies.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function B Study Section (MSFB)
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Preusch, Peter C
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Rice University
Schools of Arts and Sciences
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Sripakdeevong, Parin; Cevec, Mirko; Chang, Andrew T et al. (2014) Structure determination of noncanonical RNA motifs guided by ¹H NMR chemical shifts. Nat Methods 11:413-6
Davlieva, Milya; Donarski, James; Wang, Jiachen et al. (2014) Structure analysis of free and bound states of an RNA aptamer against ribosomal protein S8 from Bacillus anthracis. Nucleic Acids Res 42:10795-808
Thapar, Roopa; Denmon, Andria P; Nikonowicz, Edward P (2014) Recognition modes of RNA tetraloops and tetraloop-like motifs by RNA-binding proteins. Wiley Interdiscip Rev RNA 5:49-67
Chang, Andrew T; Nikonowicz, Edward P (2013) Solution NMR determination of hydrogen bonding and base pairing between the glyQS T box riboswitch Specifier domain and the anticodon loop of tRNA(Gly). FEBS Lett 587:3495-9
Chang, Andrew T; Nikonowicz, Edward P (2012) Solution nuclear magnetic resonance analyses of the anticodon arms of proteinogenic and nonproteinogenic tRNA(Gly). Biochemistry 51:3662-74
Wang, Jiachen; Nikonowicz, Edward P (2011) Solution structure of the K-turn and Specifier Loop domains from the Bacillus subtilis tyrS T-box leader RNA. J Mol Biol 408:99-117
Denmon, Andria P; Wang, Jiachen; Nikonowicz, Edward P (2011) Conformation effects of base modification on the anticodon stem-loop of Bacillus subtilis tRNA(Tyr). J Mol Biol 412:285-303
Wang, Jiachen; Henkin, Tina M; Nikonowicz, Edward P (2010) NMR structure and dynamics of the Specifier Loop domain from the Bacillus subtilis tyrS T box leader RNA. Nucleic Acids Res 38:3388-98