This proposal seeks continued support for exploration of structure and function of the ribosome actively engaged in protein synthesis, by cryo-electron microscopy and single-particle reconstruction. Several seminal discoveries regarding the dynamics of this process have been made in pursuit of this approach;among these the ratchet motion during mRNA-tRNA translocation and the spring-like deformation of the aminoacyl-tRNA as it enters the ribosome. The emphasis of the proposed studies is twofold: to characterize suitably stabilized transition states during both decoding and translocation at high resolution, and to study the time course of both processes using time-resolved cryo-electron microscopy (ms range) of pre-equilibrium samples. A novel monolithic microfluidic mixing and spraying device developed at the NCRR/NIH RVBC Resource at the Wadsworth Center in Albany will be tested and used in collaboration with the Resource, and the technology will be later transferred to the Columbia lab. Samples will be obtained from collaborators Drs. Rachel Green, Johns Hopkins University and Mans Ehrenberg, Uppsala University. Additional collaborations are in the areas of classification of heterogeneous samples and Molecular Dynamics simulations. Density maps when obtained at sufficient resolution will be analyzed by flexible fitting and interpreted in the rich context of structural, kinetics, single-molecule FRET, and biochemical data.

Public Health Relevance

The ribosome performs protein synthesis in all cells. The research proposed will further the understanding of its functional dynamics, and thus contribute to a fundamental understanding of all life processes. Specifically, understanding the function of bacterial ribosomes is furthering our ability to combat drug resistance.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM055440-17
Application #
8501509
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Flicker, Paula F
Project Start
1997-01-01
Project End
2014-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
17
Fiscal Year
2013
Total Cost
$304,919
Indirect Cost
$113,849
Name
Columbia University (N.Y.)
Department
Biochemistry
Type
Schools of Medicine
DUNS #
621889815
City
New York
State
NY
Country
United States
Zip Code
10032
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Chen, Bo; Shen, Bingxin; Frank, Joachim (2014) Particle migration analysis in iterative classification of cryo-EM single-particle data. J Struct Biol 188:267-73
Boël, Grégory; Smith, Paul C; Ning, Wei et al. (2014) The ABC-F protein EttA gates ribosome entry into the translation elongation cycle. Nat Struct Mol Biol 21:143-51
Langlois, Robert; Pallesen, Jesper; Ash, Jordan T et al. (2014) Automated particle picking for low-contrast macromolecules in cryo-electron microscopy. J Struct Biol 186:1-7
Sharma, Gyanesh; Pallesen, Jesper; Das, Sanchaita et al. (2013) Affinity grid-based cryo-EM of PKC binding to RACK1 on the ribosome. J Struct Biol 181:190-4
Frank, Joachim (2013) Story in a sample-the potential (and limitations) of cryo-electron microscopy applied to molecular machines. Biopolymers 99:832-6
Li, Wen; Atkinson, Gemma C; Thakor, Nehal S et al. (2013) Mechanism of tetracycline resistance by ribosomal protection protein Tet(O). Nat Commun 4:1477
Agirrezabala, Xabier; Liao, Hstau Y; Schreiner, Eduard et al. (2012) Structural characterization of mRNA-tRNA translocation intermediates. Proc Natl Acad Sci U S A 109:6094-9
Huang, Tao; Shaikh, Tanvir R; Gupta, Kushol et al. (2011) The group II intron ribonucleoprotein precursor is a large, loosely packed structure. Nucleic Acids Res 39:2845-54
Agirrezabala, Xabier; Schreiner, Eduard; Trabuco, Leonardo G et al. (2011) Structural insights into cognate versus near-cognate discrimination during decoding. EMBO J 30:1497-507

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