The goal of this project is to understand the molecular mechanisms of protein synthesis based on determination of x-ray crystal structures of functional complexes of ribosomes. Now that the structure of the ribosome is known at high resolution, the challenge is to determine how its structure changes in different functional states during protein synthesis. One major unsolved problem is the mechanism of coupled translocation of mRNA and tRNA, which involves large-scale rearrangements of ribosome structure, and is catalyzed by elongation factor EF-G. Complexes of ribosomes bound with EF-G will be trapped in intermediate states of translocation by use of GTP analogs, antibiotics and directed mutations in the ribosome and EF-G for crystallization and structure determination. A second project is to understand the mechanisms of termination of protein synthesis, which depends on the class I release factors RF1 and RF2, and requires participation of release factor RF3 and GTP to then release RF1 and RF2. How RF3 causes release of the type I factors is unknown, but is believed to involve changes in ribosome conformation similar to those seen for EF-G during translocation. RF3 will be bound to termination complexes containing RF1 or RF2 bound in response to a stop codon, and crystallized for structure determination. A third poorly understood mechanism is how the ribosome unwinds structured mRNAs with its endogenous helicase activity. This problem will be addressed by determining the structures of ribosomes containing mRNAs with double-helical and pseudoknot structures stalled in their helicase active sites. 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 structures of functional complexes of bacterial ribosomes will therefore 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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM059140-13
Application #
8291743
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Flicker, Paula F
Project Start
1999-04-01
Project End
2016-03-31
Budget Start
2012-05-01
Budget End
2013-03-31
Support Year
13
Fiscal Year
2012
Total Cost
$436,833
Indirect Cost
$141,251
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
Mohan, Srividya; Donohue, John Paul; Noller, Harry F (2014) Molecular mechanics of 30S subunit head rotation. Proc Natl Acad Sci U S A 111:13325-30
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
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
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
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
Korostelev, Andrei; Noller, Harry F (2007) Analysis of structural dynamics in the ribosome by TLS crystallographic refinement. J Mol Biol 373:1058-70
Korostelev, Andrei; Trakhanov, Sergei; Asahara, Haruichi et al. (2007) Interactions and dynamics of the Shine Dalgarno helix in the 70S ribosome. Proc Natl Acad Sci U S A 104:16840-3
Noller, Harry F (2004) The driving force for molecular evolution of translation. RNA 10:1833-7
Lancaster, Laura; Kiel, Michael C; Kaji, Akira et al. (2002) Orientation of ribosome recycling factor in the ribosome from directed hydroxyl radical probing. Cell 111:129-40

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