The fundamental importance of the ribosome in cellular function is evidenced by its universality and fundamental conservation throughout all forms of life. Functional complexes of the bacterial ribosome undergo large conformational changes during the course of protein synthesis. Recent dramatic progress in the elucidation of ribosome structure by both X-ray crystallography and cryoelectron microscopy has provided some of the best evidence for such changes. At the same time, detailed rate studies of initiation and elongation partial reactions, even though for the most part incomplete, have shown each of these processes to be complex, multistep reactions, raising the question of the extent to which specific structural changes can be assigned to specific steps described in proposed kinetic mechanisms. Our work has the overall goals of a) fully elucidating the kinetic mechanisms of initiation and elongation for wild-type ribosomes using fluorescence stopped-flow, quenched flow, and FRET approaches, focusing on three G- protein factors, EF-G, EF-Tu, and IF2, and the ribosomal site with which they all interact, the so-called GAC (GTPase Activation Center; and b) determining the importance of specific interactions for specific steps of ribosome catalysis by conducting structure-function analyses, measuring how specific perturbations in the protein synthesis machinery affect rates of specific steps of protein synthesis. The specific perturbations will be induced by mutation of tRNA, mutation of the peptidyl transferase center (PTC), and by addition of antibiotics targeting either the GAC (thiostrepton) or the PTC (oxazolidinones and sparsomycin). The improved understanding of ribosomal function that will result from our work could have important consequences for the design, testing, and utilization of new ribosomal antibiotics, since it should provide a paradigm for determining the specific effect(s) of a given antibiotic on ribosomal function, which can be linked directly to knowledge of the site of antibiotic binding. Medical interest in ribosomal antibiotics has been growing as bacterial resistance to beta-lactams and quinolines has become more widespread. Indeed, major pharmaceutical companies are now devoting considerable resources toward both the screening and rational design approaches for the discovery of new ribosomal antibiotics, as well as toward the modification of known antibiotics so as to overcome bacterial resistance while retaining strong inhibitory activity, largely as a result of the successful development of the new oxazolidinone family of ribosomal antibiotics and of the increased knowledge of the structure and function of the bacterial ribosome.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM071014-01
Application #
6770905
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Rhoades, Marcus M
Project Start
2004-04-01
Project End
2008-03-31
Budget Start
2004-04-01
Budget End
2005-03-31
Support Year
1
Fiscal Year
2004
Total Cost
$304,375
Indirect Cost
Name
University of Pennsylvania
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Chen, Chunlai; Stevens, Benjamin; Kaur, Jaskiran et al. (2011) Allosteric vs. spontaneous exit-site (E-site) tRNA dissociation early in protein synthesis. Proc Natl Acad Sci U S A 108:16980-5
Mikolajka, Aleksandra; Liu, Hanqing; Chen, Yuanwei et al. (2011) Differential effects of thiopeptide and orthosomycin antibiotics on translational GTPases. Chem Biol 18:589-600
Liu, Hanqing; Chen, Chunlai; Zhang, Haibo et al. (2011) The conserved protein EF4 (LepA) modulates the elongation cycle of protein synthesis. Proc Natl Acad Sci U S A 108:16223-8
Liu, Cuiping; Gamper, Howard; Liu, Hanqing et al. (2011) Potential for interdependent development of tRNA determinants for aminoacylation and ribosome decoding. Nat Commun 2:329
Kaur, Jaskiran; Raj, Monika; Cooperman, Barry S (2011) Fluorescent labeling of tRNA dihydrouridine residues: Mechanism and distribution. RNA 17:1393-400
Barhoom, Sima; Kaur, Jaskiran; Cooperman, Barry S et al. (2011) Quantitative single cell monitoring of protein synthesis at subcellular resolution using fluorescently labeled tRNA. Nucleic Acids Res 39:e129
Chen, Chunlai; Stevens, Benjamin; Kaur, Jaskarin et al. (2011) Single-molecule fluorescence measurements of ribosomal translocation dynamics. Mol Cell 42:367-77
Blanchard, Scott C; Cooperman, Barry S; Wilson, Daniel N (2010) Probing translation with small-molecule inhibitors. Chem Biol 17:633-45
Liu, Hanqing; Pan, Dongli; Pech, Markus et al. (2010) Interrupted catalysis: the EF4 (LepA) effect on back-translocation. J Mol Biol 396:1043-52
Starosta, Agata L; Qin, Haiou; Mikolajka, Aleksandra et al. (2009) Identification of distinct thiopeptide-antibiotic precursor lead compounds using translation machinery assays. Chem Biol 16:1087-96

Showing the most recent 10 out of 25 publications