The emergence of bacterial superbugs with resistance to even the most powerful antibiotics currently available poses a serious threat to human health and demands the development of new antibiotics. Approximately half of all clinically used antibiotics target the process of protein synthesis in bacteria. Protein synthesis s an essential cellular process whereby messenger RNA (mRNA) copies of the genes encoded by an organism's DNA genome are translated into their corresponding protein products by the cellular translational machinery (TM). Antibiotics that specifically target bacterial protein synthesis do so by exploiting subtle differences between the bacterial and eukaryotic TMs. The ultimate goal of this proposal is to inform antibiotic development efforts through the identificatin of new antibiotic targets within the bacterial TM. Herein, we focus on the initiation phase of translation, the step of the protein synthesis pathway where bacteria and eukaryotes differ the most. Translation initiation is a dynamic, multi-step process that, in bacteria, begins with the formation of a 30S initiation complex (30S IC) comprised of the small 30S ribosomal subunit, the mRNA to be translated, an initiator formylmethionyl-transfer RNA (tRNA), and three initiation factors (IFs). Subsequently, the large 50S ribosomal subunit joins to the 30S IC to form a functional 70S initiation complex. Subunit joining is an essential feature of this process and bacterial-specific aspects of subunit joining consequently represent viable antibiotic targets. In this proposal, we will use a combination of molecular biological, single-molecule biophysical, biochemical, and structural strategies to investigate three poorly understood aspects of the subunit joining reaction:
In Aim 1, we will investigate how the conformational dynamics of the 30S IC-bound IFs drive and regulate subunit joining;
In Aim 2, we will examine how structural rearrangements of the 30S subunit and associated changes in the positions of IF- and tRNA ligands within the 30S IC regulate subunit joining;
In Aim 3, we will study the roles that the individual components of the factor-binding site of the 50S subunit play in directing the subunit joining reaction. Our guiding hypothesis, based on extensive ensemble studies of translation initiation and accumulating single-molecule studies of the elongation phase of translation, is that the IFs, tRNA, 30S subunit, and 50S subunit stochastically fluctuate between various conformational states, some of which are conducive to subunit joining, and others that are inhibitory. In this model, shifts towards subunit joining-competent states would up-regulate protein synthesis, while shifts towards subunit joining-inhibitory states would down-regulate protein synthesis; development of small-molecule drugs designed to destabilize the competent states or stabilize the inhibitory states in a bacteria-specific manner could therefore provide a means of generating new antibiotics. The proposed studies will provide a comprehensive mechanistic understanding of the subunit joining reaction and, in doing so, will aid in the identification of novel bacteria- specific aspects of the reaction that can serve as targets for th development of next-generation antibiotics.

Public Health Relevance

The rapid emergence of multi-drug resistant bacterial pathogens poses a significant threat to human health; it is estimated that antibiotic resistant bacterial pathogens infect about 2 million people every year in the United States alone and approximately 23,000 of these people die as a result of these infections. This proposal is designed to fill a critical knowledge gap in our understanding of the mechanism through which the translation of messenger RNA into protein is initiated in bacteria, a step in the bacterial protein synthesis pathway that represents an underexploited antibiotic drug target. By uniquely employing a powerful combination of techniques, the proposed studies will enable previously underexplored aspects of translation initiation to be comprehensively investigated, thereby informing the rational design of next-generation antibiotics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM084288-08
Application #
9248369
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Preusch, Peter
Project Start
2008-12-01
Project End
2019-02-28
Budget Start
2017-03-01
Budget End
2018-02-28
Support Year
8
Fiscal Year
2017
Total Cost
$287,611
Indirect Cost
$104,735
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
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
Kinz-Thompson, Colin D; Gonzalez Jr, Ruben L (2018) Increasing the Time Resolution of Single-Molecule Experiments with Bayesian Inference. Biophys J 114:289-300
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
Greenfeld, Max; van de Meent, Jan-Willem; Pavlichin, Dmitri S et al. (2015) Single-molecule dataset (SMD): a generalized storage format for raw and processed single-molecule data. BMC Bioinformatics 16:3
Ruehle, Marisa D; Zhang, Haibo; Sheridan, Ryan M et al. (2015) A dynamic RNA loop in an IRES affects multiple steps of elongation factor-mediated translation initiation. Elife 4:
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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