title: Initial bridges between two ribosomal subunits are formed within 9.4 milliseconds, as studied by time-resolved cryo-EM.
abstract: 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.
authors: Laboratory of Cellular and Molecular Basis of Diseases, Division of Translational Medicine, Wadsworth Center, New York State Department of Health, Albany, NY 12201; shaikh@ceitec.muni.cz Rajendra.Agrawal@health.ny.gov.
authors: Tanvir R
authors: TR
authors: Shaikh
authors: Laboratory of Cellular and Molecular Basis of Diseases, Division of Translational Medicine, Wadsworth Center, New York State Department of Health, Albany, NY 12201;
authors: Aymen S
authors: AS
authors: Yassin
authors: Center for Integrated Electronics, Rensselaer Polytechnic Institute, Troy, NY 12180; and.
authors: Zonghuan
authors: Z
authors: Lu
authors: Laboratory of Cellular and Molecular Basis of Diseases, Division of Translational Medicine, Wadsworth Center, New York State Department of Health, Albany, NY 12201;
authors: David
authors: D
authors: Barnard
authors: Laboratory of Cellular and Molecular Basis of Diseases, Division of Translational Medicine, Wadsworth Center, New York State Department of Health, Albany, NY 12201;
authors: Xing
authors: X
authors: Meng
authors: Center for Integrated Electronics, Rensselaer Polytechnic Institute, Troy, NY 12180; and.
authors: Toh-Ming
authors: TM
authors: Lu
authors: Laboratory of Cellular and Molecular Basis of Diseases, Division of Translational Medicine, Wadsworth Center, New York State Department of Health, Albany, NY 12201;Department of Biomedical Sciences, School of Public Health, State University of New York at Albany, Albany, NY 12222.
authors: Terence
authors: T
authors: Wagenknecht
authors: Laboratory of Cellular and Molecular Basis of Diseases, Division of Translational Medicine, Wadsworth Center, New York State Department of Health, Albany, NY 12201;Department of Biomedical Sciences, School of Public Health, State University of New York at Albany, Albany, NY 12222 shaikh@ceitec.muni.cz Rajendra.Agrawal@health.ny.gov.
authors: Rajendra K
authors: RK
authors: Agrawal
mesh_terms: D002474:Cell-Free System; D020285:Cryoelectron Microscopy / Q000379:methods*; D008958:Models, Molecular; D012270:Ribosomes / Q000737:chemistry* / Q000648:ultrastructure
keywords: millisecond time resolution cryo-EM; ribosomal intersubunit bridges