Translation of the genetic information in the cell into functional proteins is an essential and highly conserved function. The ribosome, a two-subunit macromolecular complex, composed in bacteria of three large RNAs (rRNAs) and more than 50 proteins (r-proteins), is the catalyst and framework for the intricate and coordinated process of translation. Peptidyl transferase, likely the most primitive activity of the ribosome, is of central importance. Theoretical considerations of the origin of life predict a central role for the rRNAs in translation, a prediction strengthened by the demonstration of catalysis by RNA and consistent with the extreme phylogenetic conservation observed among rRNA sequences. Extensive biochemical and genetic studies indicate that the rRNAs play a primary functional role in the processes of translation, and in particular 23S rRNA in the central catalytic event, peptide bond formation. Structural studies of the ribosome, including cryoelectron microscopy and X-ray crystallography, are yielding a wealth of information. However, the static views of the ribosome yielded by such approaches will not reveal the mechanism or dynamics of the intricate process of translation. This proposal describes in vitro genetic and biochemical approaches to identify the molecular components at the active site of peptidyl transferase (RNA or protein). The goal of these studies is to understand how these components conspire in the formation of peptide bonds and to understand how this activity is controlled between each catalytic cycle to allow for the generation of high-fidelity encoded protein products. First, site-directed mutagenesis and modification interference approaches will be used to identify nucleotides in 23S rRNA that are specifically involved in tRNA substrate binding and catalysis by the ribosome. Second, a recently developed iterative in vitro selection approach will be used to optimize the activity of ribosomes reconstituted from in vitro transcribed Bacillus stearothermophilus 23S rRNA. Finally, a variety of biochemical approaches including crosslinking, kinetic studies and chemical modification will be used to characterize the structure and function of the hybrid state of tRNA binding in translational elongation. Information obtained from these studies will eventually help to define the features of current ribosome- specific antibiotics which make them effective and the mechanism by which these same antibiotics are evaded by the host. Ultimately, this will lead to the rational design of novel antibiotics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM059425-05S1
Application #
7022845
Study Section
Molecular Biology Study Section (MBY)
Program Officer
Rhoades, Marcus M
Project Start
1999-05-01
Project End
2005-11-30
Budget Start
2003-05-01
Budget End
2005-11-30
Support Year
5
Fiscal Year
2005
Total Cost
$77,236
Indirect Cost
Name
Johns Hopkins University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
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Buskirk, Allen R; Green, Rachel (2017) Ribosome pausing, arrest and rescue in bacteria and eukaryotes. Philos Trans R Soc Lond B Biol Sci 372:
Petropoulos, Alexandros D; McDonald, Megan E; Green, Rachel et al. (2014) Distinct roles for release factor 1 and release factor 2 in translational quality control. J Biol Chem 289:17589-96
Koutmou, Kristin S; McDonald, Megan E; Brunelle, Julie L et al. (2014) RF3:GTP promotes rapid dissociation of the class 1 termination factor. RNA 20:609-20
Guydosh, Nicholas R; Green, Rachel (2014) Dom34 rescues ribosomes in 3' untranslated regions. Cell 156:950-62
Green, Rachel; Rogers, Elizabeth J (2013) Transformation of chemically competent E. coli. Methods Enzymol 529:329-36
Buskirk, Allen R; Green, Rachel (2013) Biochemistry. Getting past polyproline pauses. Science 339:38-9
He, Shan L; Green, Rachel (2013) Northern blotting. Methods Enzymol 530:75-87
Shoemaker, Christopher J; Green, Rachel (2012) Translation drives mRNA quality control. Nat Struct Mol Biol 19:594-601
Agirrezabala, Xabier; Liao, Hstau Y; Schreiner, Eduard et al. (2012) Structural characterization of mRNA-tRNA translocation intermediates. Proc Natl Acad Sci U S A 109:6094-9

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