The long term objective ot tins project is to develop new antibacterial agents targeted against bacterial ribosome recycling factor (RRF) because RRF is an essential factor for prokaryotic translation but not for eukaryotic cytoplasmic protein synthesis. New antibacterial agents are urgently needed because of the emergence of resistant pathogens to the available antibiotics. RRF, together with elongation factor G (EF-G) or release factor 3 (RF3), catalyzes disassembly of the post-termination complex of ribosomes to recycle the ribosomes. In addition, RRF reduces translational error. RRF is a near perfect tRNA mimic. It is postulated that RRF binds to the ribosome and then translocated by EF-G on the ribosome in a fashion similar to tRNA. Inhibitors of tRNA binding to the ribo some and tRNA translocation will be used to examine this hypothesis. The interaction of RRF with ribosomes and EF-G will be studied by electron microscopy. Chemical cross-linking between RRF and the ribosome will be performed. Protection of ribosomal RNA from the effects of chemical agents by bound RRF will be examined to determine the ribosomal binding site of RRF. The possible influence of RRF on the ribosomal binding site of EF-G will also be examined. The gene encoding EF-G or RF3 will be mutagenized to explore the site of interaction with RRF. Other possible biological components which interact with RRF will be determined by identifying the intergenic suppressor genes of temperature sensitive RRF. The yeast two-hybrid system will also be utilized to find additional components that may interact with RRF. The active site of RRF will be explored using monoclonal antibodies against RRF. The mechanism of how RRF reduces translational error will be elucidated. RRF is postulated to release non-cognate or near cognate peptidyl tRNA from the ribosomal P (peptidyl)-site during the chain elongation steps. The release of cognate peptidyl tRNA from the ribosome by RRF and EF-G will be compared with that of the non- or near cognate peptidyl tRNA. At the A (acceptor) site, RRF is postulated to reduce binding of non- or near cognate aminoacyl tRNA by occupying the A site. The effect of RRF on the fidelity of aminoacyl tRNA binding will be tested. Two newly discovered inhibitors of the E. coil RRF reaction, purpuromycin and T. maritima RRF, will be studied as to their mechanism of inhibition by biochemical as well as crystallographic means

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM060429-04
Application #
6687798
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Rhoades, Marcus M
Project Start
2001-01-01
Project End
2005-12-31
Budget Start
2004-01-01
Budget End
2005-12-31
Support Year
4
Fiscal Year
2004
Total Cost
$285,300
Indirect Cost
Name
University of Pennsylvania
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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Hirokawa, Go; Iwakura, Nobuhiro; Kaji, Akira et al. (2008) The role of GTP in transient splitting of 70S ribosomes by RRF (ribosome recycling factor) and EF-G (elongation factor G). Nucleic Acids Res 36:6676-87
Pai, Raj D; Zhang, Wen; Schuwirth, Barbara S et al. (2008) Structural Insights into ribosome recycling factor interactions with the 70S ribosome. J Mol Biol 376:1334-47
Demeshkina, Natalia; Hirokawa, Go; Kaji, Akira et al. (2007) Novel activity of eukaryotic translocase, eEF2: dissociation of the 80S ribosome into subunits with ATP but not with GTP. Nucleic Acids Res 35:4597-607
Borovinskaya, Maria A; Pai, Raj D; Zhang, Wen et al. (2007) Structural basis for aminoglycoside inhibition of bacterial ribosome recycling. Nat Struct Mol Biol 14:727-32
Barat, Chandana; Datta, Partha P; Raj, V Samuel et al. (2007) Progression of the ribosome recycling factor through the ribosome dissociates the two ribosomal subunits. Mol Cell 27:250-61
Hirokawa, Go; Demeshkina, Natalia; Iwakura, Nobuhiro et al. (2006) The ribosome-recycling step: consensus or controversy? Trends Biochem Sci 31:143-9
Raj, V Samuel; Kaji, Hideko; Kaji, Akira (2005) Interaction of RRF and EF-G from E. coli and T. thermophilus with ribosomes from both origins--insight into the mechanism of the ribosome recycling step. RNA 11:275-84
Hirokawa, Go; Nijman, Romana M; Raj, V Samuel et al. (2005) The role of ribosome recycling factor in dissociation of 70S ribosomes into subunits. RNA 11:1317-28
Agrawal, Rajendra K; Sharma, Manjuli R; Kiel, Michael C et al. (2004) Visualization of ribosome-recycling factor on the Escherichia coli 70S ribosome: functional implications. Proc Natl Acad Sci U S A 101:8900-5

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