The ribosome, a large ribonucleoprotein enzyme, is universally responsible for translating messenger RNAs (mRNAs) into the encoded protein products. This process is among one of the most fundamental and highly regulated in all living things. Recent advances in the structural biology of protein synthesis have provided atomic resolution structures of the ribosome as well as lower-resolution snapshots of ribosomal complexes trapped in the process of translation. What is currently lacking from mechanistic models of ribosome function is a description of the kinetics governing transitions from one conformational state of the ribosome to the next. Although difficult, and often impossible, to study precisely using bulk biochemical methods, these conformational dynamics have been shown to be of prime importance in translation. The initiation phase of protein synthesis is the focal point for the translational control of gene expression. As such, the initiation pathway serves as a very effective target for small molecule antibiotics, human viral pathogens, and deregulation of initiation is increasingly causally linked to tumorigenesis. The initiation reaction is an amazingly dynamic process, involving the interaction of numerous translation initiation factors (IFs) with the ribosome in a highly-coordinated and specific series of molecular events. We hypothesize that IFs regulate the initiation pathway by precisely altering the stabilities of dynamically heterogeneous conformational intermediates of the initiation machinery. To address this dynamic conformational heterogeneity, we will use single-molecule fluorescence resonance energy transfer (smFRET). smFRET provides a unique tool for characterizing the conformational dynamics of individual molecules, eliminating the population averaging inherent in ensemble studies and revealing the dynamic heterogeneity of the system. These data will help elucidate the basic mechanism of translation initiation, providing crucial kinetic information that has heretofore remained inaccessible in bulk studies. Specifically, we will use these techniques to (1) investigate the dynamics of initiation factor 2 (IF2) and initiator transfer RNA (tRNAi) that regulate tRNAi selection during initiation, (2) determine how coupling of ribosome and tRNAi conformational dynamics control the fidelity of tRNAi and start codon selection, and (3) establish the currently unknown mechanism through which initiation factor 3 (IF3) acts to proofread the fidelity of the initiation reaction. Our ability to correlate the kinetics of critical conformational changes with fundamental biochemical steps in the initiation pathway will aid the development of a complete mechanistic model for this universal and biomedically relevant biological process.

Public Health Relevance

Protein synthesis, catalyzed in all cells by an enzyme called the ribosome, is an important focal point for the control of gene expression. Loss of control over the initiation step of protein synthesis is induced by antibiotic drugs, is exploited by human viral pathogens, and is implicated in cancer. This proposal uses state-of-the-art microscopic technologies to addresses fundamental aspects of initiation that hold great promise towards revealing how this step in gene expression is controlled and how that control is exploited in disease.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function B Study Section (MSFB)
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Preusch, Peter C
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Columbia University (N.Y.)
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New York
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Subramanyam, Shyamal; Kinz-Thompson, Colin D; Gonzalez Jr, Ruben L et al. (2018) Observation and Analysis of RAD51 Nucleation Dynamics at Single-Monomer Resolution. Methods Enzymol 600:201-232
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Caban, Kelvin; Pavlov, Michael; Ehrenberg, Måns et al. (2017) A conformational switch in initiation factor 2 controls the fidelity of translation initiation in bacteria. Nat Commun 8:1475
Kinz-Thompson, C D; Bailey, N A; Gonzalez Jr, R L (2016) Precisely and Accurately Inferring Single-Molecule Rate Constants. Methods Enzymol 581:187-225
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Caban, Kelvin; Gonzalez Jr, Ruben L (2015) The emerging role of rectified thermal fluctuations in initiator aa-tRNA- and start codon selection during translation initiation. Biochimie 114:30-8
Thompson, Colin D Kinz; Sharma, Ajeet K; Frank, Joachim et al. (2015) Quantitative Connection between Ensemble Thermodynamics and Single-Molecule Kinetics: A Case Study Using Cryogenic Electron Microscopy and Single-Molecule Fluorescence Resonance Energy Transfer Investigations of the Ribosome. J Phys Chem B 119:10888-10901
Wang, Jiangning; Caban, Kelvin; Gonzalez Jr, Ruben L (2015) Ribosomal initiation complex-driven changes in the stability and dynamics of initiation factor 2 regulate the fidelity of translation initiation. J Mol Biol 427:1819-34
Pereira, Sandro F F; Gonzalez Jr, Ruben L; Dworkin, Jonathan (2015) Protein synthesis during cellular quiescence is inhibited by phosphorylation of a translational elongation factor. Proc Natl Acad Sci U S A 112:E3274-81

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