The ribosome is a large complex of ribonucleoproteins, which interacts with several protein factors and RNA molecules to conduct the life sustaining process of protein biosynthesis. It is also the target site of several antibiotics, which inhibit specific steps during the interaction of protein factors with the ribosome. Very little is known about molecular details of the dynamic events that take place during this interaction of ribosomes. Very recently, impressive progress has been made towards solving the atomic structure (for example, structures of ribosomal subunits from a thermophilic bacterium and a halophilic archaeon are known to 3 - 2.4 Angstrom units resolution); however, it will take many years before various dynamic events can be trapped and analyzed by X-ray crystallography of the complete ribosome. This proposal describes the development of a method that is uniquely suited to study the dynamic events on the ribosome at near atomic resolution and in close to in vivo condition. We plan to strategically label specific amino acid residues of protein ligands (EF-G, EF-Tu, release and recycling factors) with heavy metal clusters and form a complex with the ribosome. The complex will be imaged by the technique of single particle reconstruction of images obtained from cryo-electron microscopy (conceived and successfully applied in the laboratory of Dr. Joachim Frank, the co-PI). The resulting density maps will be analyzed in terms of the X-ray structure of the ligand by a flexible docking method. We have preliminary data showing that the technique of labeling of specific amino acid with heavy metal and binding of the heavy-metal-labeled ligand to the ribosome are feasible. Although the approach has broad applications, the main emphasis of this proposal is to understand the molecular basis of the conformational changes of various ribosome-binding factors and their relevance in protein biosynthesis.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Biophysical Chemistry Study Section (BBCB)
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Deatherage, James F
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Wadsworth Center
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Li, Yunlong; Sharma, Manjuli R; Koripella, Ravi K et al. (2018) Zinc depletion induces ribosome hibernation in mycobacteria. Proc Natl Acad Sci U S A 115:8191-8196
Agrawal, Rajendra Kumar; Wang, Hong-Wei; Belfort, Marlene (2016) Forks in the tracks: Group II introns, spliceosomes, telomeres and beyond. RNA Biol 13:1218-1222
Qu, Guosheng; Kaushal, Prem Singh; Wang, Jia et al. (2016) Structure of a group II intron in complex with its reverse transcriptase. Nat Struct Mol Biol 23:549-57
Kaushal, Prem S; Sharma, Manjuli R; Agrawal, Rajendra K (2015) The 55S mammalian mitochondrial ribosome and its tRNA-exit region. Biochimie 114:119-26
Lu, Zonghuan; Barnard, David; Shaikh, Tanvir R et al. (2014) Gas-Assisted Annular Microsprayer for Sample Preparation for Time-Resolved Cryo-Electron Microscopy. J Micromech Microeng 24:115001
Chen, Eileen; Sharma, Manjuli R; Shi, Xinying et al. (2014) Fragile X mental retardation protein regulates translation by binding directly to the ribosome. Mol Cell 54:407-417
Shaikh, Tanvir R; Yassin, Aymen S; Lu, Zonghuan et al. (2014) Initial bridges between two ribosomal subunits are formed within 9.4 milliseconds, as studied by time-resolved cryo-EM. Proc Natl Acad Sci U S A 111:9822-7
Kaushal, Prem S; Sharma, Manjuli R; Booth, Timothy M et al. (2014) Cryo-EM structure of the small subunit of the mammalian mitochondrial ribosome. Proc Natl Acad Sci U S A 111:7284-9
Yokoyama, Takeshi; Shaikh, Tanvir R; Iwakura, Nobuhiro et al. (2012) Structural insights into initial and intermediate steps of the ribosome-recycling process. EMBO J 31:1836-46
Agrawal, Rajendra K; Sharma, Manjuli R (2012) Structural aspects of mitochondrial translational apparatus. Curr Opin Struct Biol 22:797-803

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