This proposal seeks continued support for exploration of structure and function of the ribosome actively engaged in protein synthesis, by cryo-electron microscopy (cryo-EM) and single-particle reconstruction. Protein biosynthesis is one of the most fundamental processes of life, but despite seminal work in solving the structure of the ribosome, this process is not well understood. Cryo-EM is uniquely suited to capture the molecule in functional states under close to native conditions. In this lab, several seminal discoveries regarding the dynamics of this process in both eubacterial and eukaryotic translation have been made in the past;among these the ratchet-like motion during mRNA-tRNA translocation and the large spring-like deformation of the aminoacyl-tRNA as it enters the ribosome during the aminoacyl-tRNA selection process. Special focus of this research program will be the eukaryotic (yeast and mammalian) ribosome.
The aims of the proposed studies are threefold: (1) to make use of tilt pair recoding, novel technology for online data capture in the electron microscope, workflow optimization, and availability of a Titan Krios instrument at Janelia Farm, and other improvements in order to achieve atomic (better than 3.5A) resolution for selected ribosome complexes of special interest, such as ribosomes of trypanosomes and a yeast translocation complex;(2) to study the process of mammalian eukaryotic translation initiation by cryo-EM visualization of selected initiation complexes;(3) to study, with the aid of advanced classification methods, the distribution of states and their structural manifestations in a freely equilibrating, factor-free, eukaryotic ribosome sample as a function of relevant parameters such as temperature and ionic conditions. Samples will be obtained from several collaborating labs that are specialized in eukaryotic translation. Additional important collaborations are in the area of eukaryotic ribosome structure, smFRET, classification of heterogeneous samples by manifold embedding, and atomic modeling. Density maps when obtained at sufficient resolution will be analyzed by flexible fitting and interpreted in the rich context of existing structural, kinetics, single-molecule FRET, and biochemical data.

Public Health Relevance

The ribosome performs protein synthesis in all cells in all life forms. The research proposed will further the understanding of its functional dynamics, and thus contribute to a fundamental understanding of all life processes. Understanding the function of the eukaryotic ribosome on a fundamental level is advancing our ability to combat debilitating human diseases arising from dysfunctions of translation, and viral takeover of the ribosome by the IRES mechanism (e.g., in hepatitis C). This knowledge also helps in developing drugs to fight eukaryotic parasites.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM029169-30A1
Application #
8369343
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Flicker, Paula F
Project Start
1982-04-01
Project End
2016-05-31
Budget Start
2012-09-01
Budget End
2013-05-31
Support Year
30
Fiscal Year
2012
Total Cost
$548,743
Indirect Cost
$174,842
Name
Columbia University (N.Y.)
Department
Biochemistry
Type
Schools of Medicine
DUNS #
621889815
City
New York
State
NY
Country
United States
Zip Code
10032
des Georges, Amedee; Hashem, Yaser; Unbehaun, Anett et al. (2014) Structure of the mammalian ribosomal pre-termination complex associated with eRF1.eRF3.GDPNP. Nucleic Acids Res 42:3409-18
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
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
Hashem, Yaser; des Georges, Amedee; Dhote, Vidya et al. (2013) Structure of the mammalian ribosomal 43S preinitiation complex bound to the scanning factor DHX29. Cell 153:1108-19
Hashem, Yaser; des Georges, Amedee; Dhote, Vidya et al. (2013) Hepatitis-C-virus-like internal ribosome entry sites displace eIF3 to gain access to the 40S subunit. Nature 503:539-43
Hashem, Yaser; des Georges, Amedee; Fu, Jie et al. (2013) High-resolution cryo-electron microscopy structure of the Trypanosoma brucei ribosome. Nature 494:385-9
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

Showing the most recent 10 out of 109 publications