Title
Co-temporal Force and Fluorescence Measurements Reveal a Ribosomal Gear Shift Mechanism of Translation Regulation by Structured mRNAs
Date Issued
05 September 2019
Access level
open access
Resource Type
journal article
Author(s)
Desai V.P.
Frank F.
Lee A.
Righini M.
Lancaster L.
Noller H.F.
Tinoco I.
University of California
Publisher(s)
Cell Press
Abstract
The movement of ribosomes on mRNA is often interrupted by secondary structures that present mechanical barriers and play a central role in translation regulation. We investigate how ribosomes couple their internal conformational changes with the activity of translocation factor EF-G to unwind mRNA secondary structures using high-resolution optical tweezers with single-molecule fluorescence capability. We find that hairpin opening occurs during EF-G-catalyzed translocation and is driven by the forward rotation of the small subunit head. Modulating the magnitude of the hairpin barrier by force shows that ribosomes respond to strong barriers by shifting their operation to an alternative 7-fold-slower kinetic pathway prior to translocation. Shifting into a slow gear results from an allosteric switch in the ribosome that may allow it to exploit thermal fluctuations to overcome mechanical barriers. Finally, we observe that ribosomes occasionally open the hairpin in two successive sub-codon steps, revealing a previously unobserved translocation intermediate.
Start page
1007
End page
1019.e5
Volume
75
Issue
5
Language
English
OCDE Knowledge area
Biología celular, Microbiología
Bioquímica, Biología molecular
Subjects
Scopus EID
2-s2.0-85071537661
PubMed ID
Source
Molecular Cell
ISSN of the container
10972765
Sponsor(s)
Dr. Ignacio Tinoco Jr. passed away on November 15, 2016. We thank Thy Minh Thy Pham and William John van Patten for their help in the preparation of reagents; Charles E. Wickersham, Prof. Matthew Comstock, and Prof. Taepjik Ha for their help in building the instrument; and Lisa Alexander, Liang Meng Wee, Maya Sen, and Shannon Yan for useful discussions. This research was supported by the Howard Hughes Medical Institute (instrumentation and ribosome biochemistry); by NIH grants R01GM071552 and R01GM032543 (fluorescence experiments); and the Nanomachine program (KC1203), funded by the Office of Basic Energy Sciences of the U.S. Department of Energy (DOE) contract no. DE-AC02-05CH11231 (data analysis algorithms). F.F. acknowledges support from an EMBO postdoctoral fellowship and M.R. acknowledges support from the Damon Runyon Cancer Research Foundation (DRG-2096-11). V.P.D. F.F. M.R. L.L. H.F.N. I.T. and C.B. designed the study. V.P.D. F.F. and L.L. prepared all of the reagents. M.R. assembled the instrument. V.P.D. F.F. and M.R. collected the data. A.L. and V.P.D. designed and implemented the data analysis procedures. V.P.D. A.L. and F.F. analyzed the data. V.P.D. F.F. A.L. L.L. H.F.N. and C.B. wrote the manuscript. The authors declare no competing interests.
National Institutes of Health KC1203, R01GM071552 NIH
National Institute of General Medical Sciences R37GM032543 NIGMS
Irene W. and C.B. Pennington Foundation
Sources of information:
Directorio de Producción Científica
Scopus