Key Intermediates in Ribosome Recycling Visualized by Time-Resolved Cryoelectron Microscopy.
Upon encountering a stop codon on mRNA, polypeptide synthesis on the ribosome is terminated by release factors, and the ribosome complex, still bound with mRNA and P-site-bound tRNA (post-termination complex, PostTC), is split into ribosomal subunits, ready for a new round of translational initiation. Separation of post-termination ribosomes into subunits, or "ribosome recycling," is promoted by the joint action of ribosome-recycling factor (RRF) and elongation factor G (EF-G) in a guanosine triphosphate (GTP) hydrolysis-dependent manner. Here we used a mixing-spraying-based method of time-resolved cryo-electron microscopy (cryo-EM) to visualize the short-lived intermediates of the recycling process. The two complexes that contain (1) both RRF and EF-G bound to the PostTC or (2) deacylated tRNA bound to the 30S subunit are of particular interest. Our observations of the native form of these complexes demonstrate the strong potential of time-resolved cryo-EM for visualizing previously unobservable transient structures.
Integrated Program in Cellular, Molecular, and Biomedical Studies, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.
Ziao
Z
Fu
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10027, USA.
Sandip
S
Kaledhonkar
Department of Cell and Molecular Biology, Uppsala University, Uppsala 751 24, Sweden.
Anneli
A
Borg
Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
Ming
M
Sun
Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
Bo
B
Chen
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10027, USA; Howard Hughes Medical Institute, Columbia University, New York, NY 10032, USA.
Robert A
RA
Grassucci
Department of Cell and Molecular Biology, Uppsala University, Uppsala 751 24, Sweden.
Måns
M
Ehrenberg
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10027, USA; Department of Biological Sciences, Columbia University, New York, NY 10027, USA; Howard Hughes Medical Institute, Columbia University, New York, NY 10032, USA. Electronic address: jf2192@cumc.columbia.edu.
Joachim
J
Frank
D001665:Binding Sites; D020285:Cryoelectron Microscopy; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008958:Models, Molecular; D020653:Peptide Elongation Factor G / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D014176:Protein Biosynthesis; D012343:RNA, Transfer / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
D029968:Escherichia coli Proteins; D020653:Peptide Elongation Factor G; D012269:Ribosomal Proteins; C087267:ribosome releasing factor; D006160:Guanosine Triphosphate; D012343:RNA, Transfer
cryo-EM; mixing-spraying; recycling; ribosome; time-resolved