The ultimate goal of this project is to understand the molecular mechanisms of protein synthesis. The wealth of structural information obtained from cryoEM reconstructions and x-ray crystallography in recent years has provided a strong basis for studying the molecular basis of ribosome function and has led to proposal of numerous mechanistic models. However, it is essential to subject such structure-based models to experimental test. Moreover, the realization that ribosomes are highly dynamic structures, whose functional capabilities are based on both large- and small-scale molecular movements, requires experimental approaches that will be able to correlate specific dynamic events with function. This project will investigate the mechanisms of crucial steps in translation, in particulr, steps where ribosome structural dynamics play an important role and including experiments specifically designed to critically test mechanisms based on structural observations. The experimental approaches are based on biochemical and biophysical solution methods, including in vitro testing of specific functions in mutationally altered ribosomes, bulk and single-molecule FRET approaches and the use of optical tweezers. The principle aims are to investigate (i) the molecular basis of ribosomal translocation;(ii) the mechanism of translation termination;and (iii the ribosomal mRNA helicase and measurement of ribosomal forces using optical tweezers. The proposed research is of strong clinical relevance because bacterial ribosomes are a major target for numerous antibiotics. Elucidation of the structures of functional complexes of bacterial ribosomes will provide a rigorous basis for development of novel antibiotics to address the crisis of emerging drug-resistant strains of bacterial pathogens. Studying the mechanism of action of the ribosomal helicase will help to understand how retroviruses, including HIV, use pseudoknots to create programmed frame-shifting during their infectious cycle, providing clues to the design of anti-retroviral therapeutics.

Public Health Relevance

This research is of strong clinical relevance because bacterial ribosomes are a major target for numerous antibiotics. Elucidation of the mechanisms of action of bacterial ribosomes, together with knowledge of their high-resolution structures, will therefore provide a rigorous basis for development of novel antibiotics to address the crisis of emerging drug-resistant strains of bacterial pathogens. Studies on the mechanism of action of the ribosomal helicase will help to understand how retroviruses, including HIV, use pseudoknots to create programmed frame-shifting during their infectious cycle, providing clues to the design of anti-retroviral therapeutics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM017129-42
Application #
8291732
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Bender, Michael T
Project Start
1977-05-01
Project End
2016-04-30
Budget Start
2012-05-25
Budget End
2013-04-30
Support Year
42
Fiscal Year
2012
Total Cost
$442,197
Indirect Cost
$146,661
Name
University of California Santa Cruz
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
125084723
City
Santa Cruz
State
CA
Country
United States
Zip Code
95064
Zhou, Jie; Lancaster, Laura; Donohue, John Paul et al. (2014) How the ribosome hands the A-site tRNA to the P site during EF-G-catalyzed translocation. Science 345:1188-91
Liu, Tingting; Kaplan, Ariel; Alexander, Lisa et al. (2014) Direct measurement of the mechanical work during translocation by the ribosome. Elife 3:e03406
Santos, Natalia; Zhu, Jianyu; Donohue, John Paul et al. (2013) Crystal structure of the 70S ribosome bound with the Q253P mutant form of release factor RF2. Structure 21:1258-63
Ermolenko, Dmitri N; Cornish, Peter V; Ha, Taekjip et al. (2013) Antibiotics that bind to the A site of the large ribosomal subunit can induce mRNA translocation. RNA 19:158-66
Zhou, Jie; Lancaster, Laura; Donohue, John Paul et al. (2013) Crystal structures of EF-G-ribosome complexes trapped in intermediate states of translocation. Science 340:1236086
Noller, Harry F (2012) Evolution of protein synthesis from an RNA world. Cold Spring Harb Perspect Biol 4:a003681
Zhu, Jianyu; Korostelev, Andrei; Costantino, David A et al. (2011) Crystal structures of complexes containing domains from two viral internal ribosome entry site (IRES) RNAs bound to the 70S ribosome. Proc Natl Acad Sci U S A 108:1839-44
Ermolenko, Dmitri N; Noller, Harry F (2011) mRNA translocation occurs during the second step of ribosomal intersubunit rotation. Nat Struct Mol Biol 18:457-62
Cornish, Peter V; Ermolenko, Dmitri N; Staple, David W et al. (2009) Following movement of the L1 stalk between three functional states in single ribosomes. Proc Natl Acad Sci U S A 106:2571-6
Cornish, Peter V; Ermolenko, Dmitri N; Noller, Harry F et al. (2008) Spontaneous intersubunit rotation in single ribosomes. Mol Cell 30:578-88

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