Initial bridges between two ribosomal subunits are formed within 9.4 milliseconds, as studied by time-resolved cryo-EM

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Authors

SHAIKH Tanvir YASSIN A.S. LU Z.H. BARNARD D. MENG X. LU T.M. WAGENKNECHT T. AGRAWAL R. K.

Year of publication 2014
Type Article in Periodical
Magazine / Source Proceedings of the National Academy of Sciences of the United States of America
MU Faculty or unit

Central European Institute of Technology

Citation
Web http://www.pnas.org/content/111/27/9822.full.pdf+html
Doi http://dx.doi.org/10.1073/pnas.1406744111
Field Biochemistry
Keywords ribosomal intersubunit bridges; millisecond time resolution cryo-EM
Attached files
Description Association of the two ribosomal subunits during the process of translation initiation is a crucial step of protein synthesis. The two subunits (30S and 50S) of the bacterial 70S ribosome are held together by 12 dynamic bridges involving RNA-RNA, RNA-protein, and protein-protein interactions. The process of bridge formation, such as whether all these bridges are formed simultaneously or in a sequential order, is poorly understood. To understand such processes, we have developed and implemented a class of microfluidic devices that mix two components to completion within 0.4 ms and spray the mixture in the form of microdroplets onto an electron microscopy grid, yielding a minimum reaction time of 9.4 ms before cryofixation. Using these devices, we have obtained cryo-EM data corresponding to reaction times of 9.4 and 43 ms and have determined 3D structures of ribosomal subunit association intermediates. Molecular analyses of the cryo-EM maps reveal that eight intersubunit bridges (bridges B1a, B1b, B2a, B2b, B3, B7a, B7b, and B8) form within 9.4 ms, whereas the remaining four bridges (bridges B2c, B4, B5, and B6) take longer than 43 ms to form, suggesting that bridges are formed in a stepwise fashion. Our approach can be used to characterize sequences of various dynamic functional events on complex macromolecular assemblies such as ribosomes.
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