The long term objective of this grant proposal is to understand how the ribosome functions at the atomic level during protein synthesis. Our approach includes developing genetic and biochemical systems in the extreme thermophile Thermus thermophilus to construct and isolate mutants in rRNA, ribosomal proteins and translation factors.
Our first aim i s to characterize wild-type T. thermophilus ribosomes in solution by structure probing and determine modified residues by mass spectrometry. This will serve as the baseline for subsequent analysis of mutants in rRNA and ribosomal proteins. Selected mutants will be identified for crystallization attempts.
A second aim i s to investigate the nature of interactions between the ribosome and elongation factors EF-Tu and EF-G during accommodation and translocation. Selected functional sites in both subunits will be probed. A model for conformational change during translocation will be evaluated as will the mechanism of translocation inhibition by tuberactinomycin antibiotics.
A third aim will probe our collection of mutants in the peptidyltransferase active site of 23S rRNA of T. thermophilus to characterize functions in addition to peptide bond formation associated with this region, including translocation, decoding and peptide release. Fast kinetic experiments will be used to evaluate the mutants and test a model of accommodation, and the peptide antibiotic negamycin will be examined for its role in termination. These experiments should provide fundamental insights into ribosome structure-function relationships, different conformational states of the ribosome during translation and the mode of action of and resistance to clinically useful antibiotics.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics A Study Section (MGA)
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Bender, Michael T
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Brown University
Schools of Medicine
United States
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Carr, Jennifer F; Danziger, Michael E; Huang, Athena L et al. (2015) Engineering the genome of Thermus thermophilus using a counterselectable marker. J Bacteriol 197:1135-44
Carr, Jennifer F; Gregory, Steven T; Dahlberg, Albert E (2015) Transposon mutagenesis of the extremely thermophilic bacterium Thermus thermophilus HB27. Extremophiles 19:221-8
Demirci, Hasan; Murphy 4th, Frank V; Murphy, Eileen L et al. (2014) Structural analysis of base substitutions in Thermus thermophilus 16S rRNA conferring streptomycin resistance. Antimicrob Agents Chemother 58:4308-17
Demirci, Hasan; Murphy 4th, Frank; Murphy, Eileen et al. (2013) A structural basis for streptomycin-induced misreading of the genetic code. Nat Commun 4:1355
Demirci, Hasan; Wang, Leyi; Murphy 4th, Frank V et al. (2013) The central role of protein S12 in organizing the structure of the decoding site of the ribosome. RNA 19:1791-801
Demirci, Hasan; Sierra, Raymond G; Laksmono, Hartawan et al. (2013) Serial femtosecond X-ray diffraction of 30S ribosomal subunit microcrystals in liquid suspension at ambient temperature using an X-ray free-electron laser. Acta Crystallogr Sect F Struct Biol Cryst Commun 69:1066-9
Cantara, William A; Murphy 4th, Frank V; Demirci, Hasan et al. (2013) Expanded use of sense codons is regulated by modified cytidines in tRNA. Proc Natl Acad Sci U S A 110:10964-9
Monshupanee, Tanakarn; Johansen, Shanna K; Dahlberg, Albert E et al. (2012) Capreomycin susceptibility is increased by TlyA-directed 2'-O-methylation on both ribosomal subunits. Mol Microbiol 85:1194-203
Demirci, Hasan; Murphy 4th, Frank; Belardinelli, Riccardo et al. (2010) Modification of 16S ribosomal RNA by the KsgA methyltransferase restructures the 30S subunit to optimize ribosome function. RNA 16:2319-24
Demirci, Hasan; Larsen, Line H G; Hansen, Trine et al. (2010) Multi-site-specific 16S rRNA methyltransferase RsmF from Thermus thermophilus. RNA 16:1584-96

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