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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM084288-05
Application #
8399087
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Preusch, Peter C
Project Start
2008-12-01
Project End
2014-11-30
Budget Start
2012-12-01
Budget End
2014-11-30
Support Year
5
Fiscal Year
2013
Total Cost
$308,393
Indirect Cost
$114,504
Name
Columbia University (N.Y.)
Department
Chemistry
Type
Other Domestic Higher Education
DUNS #
049179401
City
New York
State
NY
Country
United States
Zip Code
10027
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Chen, Bo; Boël, Grégory; Hashem, Yaser et al. (2014) EttA regulates translation by binding the ribosomal E site and restricting ribosome-tRNA dynamics. Nat Struct Mol Biol 21:152-9
Kinz-Thompson, Colin D; Gonzalez Jr, Ruben L (2014) smFRET studies of the 'encounter' complexes and subsequent intermediate states that regulate the selectivity of ligand binding. FEBS Lett 588:3526-38
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Kim, Hee-Kyung; Liu, Fei; Fei, Jingyi et al. (2014) A frameshifting stimulatory stem loop destabilizes the hybrid state and impedes ribosomal translocation. Proc Natl Acad Sci U S A 111:5538-43
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Kinz-Thompson, Colin D; Palma, Matteo; Pulukkunat, Dileep K et al. (2013) Robustly passivated, gold nanoaperture arrays for single-molecule fluorescence microscopy. ACS Nano 7:8158-66
Elvekrog, Margaret M; Gonzalez Jr, Ruben L (2013) Conformational selection of translation initiation factor 3 signals proper substrate selection. Nat Struct Mol Biol 20:628-33
Fei, Jingyi; Richard, Arianne C; Bronson, Jonathan E et al. (2011) Transfer RNA-mediated regulation of ribosome dynamics during protein synthesis. Nat Struct Mol Biol 18:1043-51

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