Protein synthesis by the ribosome directly couples genotype to phenotype in the cell, and its regulation is central to cellular physiology. Although many steps of translation have diverged since the last common ancestor, translation initiation and ribosome recycling remain intimately coupled in both bacteria and humans. These coupled events in the translation cycle are the focus of the present application. Our understanding of the molecular mechanism of protein synthesis has undergone a revolution in the last decade, built on rapid advances in the structural biology of the ribosome. High-resolution structures are now available for the entire ribosome in both bacteria and eukaryotes, and of the large ribosomal subunit in archaea. However, important questions relating to dynamic events in translation remain unanswered due to the challenge of isolating structural intermediates. In this application, we propose to probe the molecular mechanism of ribosome recycling in bacteria, a process catalyzed by the GTPase elongation factor G and a validated target of antibiotics. We will build on our prior groundbreaking results in this area by combining x-ray crystallography, cryo-electron microscopy (cryo-EM) and single-molecule biophysics. By using x-ray crystallography, cryo-EM, and new methods and tools we have developed to study human translation initiation factor eIF3, we will also probe human translation initiation, one of the most dynamic steps in protein synthesis. We will use our unique system to functionally reconstitute human translation initiation factor eIF3, cryo-EM, genetics in Neurospora crassa, and revolutionary new methods in genome engineering to dissect the contribution of eIF3 to start codon selection on human cellular mRNAs. The combination of eIF3 reconstitution in E. coli, genetics in N. crassa, mutagenesis in human cells, and cutting-edge cryo-EM provides a powerful and unique means to unravel the molecular contributions of eIF3 to translation initiation in humans. Taken together, the three aims of this application build on the fundamental insights into ribosome structure and function obtained in the prior funding period, and address key mechanisms in translational control that could be exploited for the development of new antibiotics and therapeutics.

Public Health Relevance

Understanding how the ribosome functions in protein synthesis, or translation, is an important goal of basic research. This research has wide applications in medicine and biotechnology in the development of new antibiotics and therapeutics. Bacterial ribosomes are the target of many classes of antibiotics, and the control of human translation is important in normal development and disease prevention. Our studies will determine how dynamic events in protein synthesis dictate when and how proteins are made and will reveal new ways that translation might be manipulated to improve human health.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function C Study Section (MSFC)
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Bender, Michael T
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University of California Berkeley
Schools of Arts and Sciences
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Olivier, Nelson B; Altman, Roger B; Noeske, Jonas et al. (2014) Negamycin induces translational stalling and miscoding by binding to the small subunit head domain of the Escherichia coli ribosome. Proc Natl Acad Sci U S A 111:16274-9
Lintner, Nathanael G; Cate, Jamie H D (2014) Regulating the ribosome: a spotlight on RNA dark matter. Mol Cell 54:1-2
Noeske, Jonas; Huang, Jian; Olivier, Nelson B et al. (2014) Synergy of streptogramin antibiotics occurs independently of their effects on translation. Antimicrob Agents Chemother 58:5269-79
Smith, M Duane; Gu, Yu; Querol-Audí, Jordi et al. (2013) Human-like eukaryotic translation initiation factor 3 from Neurospora crassa. PLoS One 8:e78715
Whitford, Paul C; Blanchard, Scott C; Cate, Jamie H D et al. (2013) Connecting the kinetics and energy landscape of tRNA translocation on the ribosome. PLoS Comput Biol 9:e1003003
Pulk, Arto; Cate, Jamie H D (2013) Control of ribosomal subunit rotation by elongation factor G. Science 340:1235970
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Dunkle, Jack A; Cate, Jamie H D (2010) Ribosome structure and dynamics during translocation and termination. Annu Rev Biophys 39:227-44
Cate, Jamie H D (2010) Some reassembly required. Mol Microbiol 75:793-4
Dunkle, Jack A; Xiong, Liqun; Mankin, Alexander S et al. (2010) Structures of the Escherichia coli ribosome with antibiotics bound near the peptidyl transferase center explain spectra of drug action. Proc Natl Acad Sci U S A 107:17152-7

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