A trailing ribosome speeds up RNA polymerase at the expense of transcript fidelity via force and allostery

Liang Meng Wee; Alexander B Tong; Alfredo Jose Florez Ariza; Cristhian Cañari-Chumpitaz; Patricia Grob; Eva Nogales; Carlos J Bustamante

doi:10.1016/j.cell.2023.02.008

A trailing ribosome speeds up RNA polymerase at the expense of transcript fidelity via force and allostery

Cell. 2023 Mar 16;186(6):1244-1262.e34. doi: 10.1016/j.cell.2023.02.008.

Authors

Liang Meng Wee¹, Alexander B Tong², Alfredo Jose Florez Ariza³, Cristhian Cañari-Chumpitaz⁴, Patricia Grob⁵, Eva Nogales⁶, Carlos J Bustamante⁷

Affiliations

¹ QB3-Berkeley, Berkeley, CA, USA; Howard Hughes Medical Institute, University of California Berkeley, Berkeley, CA, USA.
² QB3-Berkeley, Berkeley, CA, USA; Department of Chemistry, University of California Berkeley, Berkeley, CA, USA.
³ QB3-Berkeley, Berkeley, CA, USA; Biophysics Graduate Group, University of California Berkeley, Berkeley, CA, USA.
⁴ QB3-Berkeley, Berkeley, CA, USA; Department of Chemistry, University of California Berkeley, Berkeley, CA, USA; Howard Hughes Medical Institute, University of California Berkeley, Berkeley, CA, USA.
⁵ QB3-Berkeley, Berkeley, CA, USA; Howard Hughes Medical Institute, University of California Berkeley, Berkeley, CA, USA; Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA.
⁶ QB3-Berkeley, Berkeley, CA, USA; Biophysics Graduate Group, University of California Berkeley, Berkeley, CA, USA; Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA; Howard Hughes Medical Institute, University of California Berkeley, Berkeley, CA, USA; Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. Electronic address: enogales@lbl.gov.
⁷ QB3-Berkeley, Berkeley, CA, USA; Biophysics Graduate Group, University of California Berkeley, Berkeley, CA, USA; Department of Chemistry, University of California Berkeley, Berkeley, CA, USA; Department of Physics, University of California Berkeley, Berkeley, CA, USA; Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA; Kavli Energy Nanoscience Institute, Berkeley, CA, USA; Howard Hughes Medical Institute, University of California Berkeley, Berkeley, CA, USA; Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. Electronic address: carlosb@berkeley.edu.

Abstract

In prokaryotes, translation can occur on mRNA that is being transcribed in a process called coupling. How the ribosome affects the RNA polymerase (RNAP) during coupling is not well understood. Here, we reconstituted the E. coli coupling system and demonstrated that the ribosome can prevent pausing and termination of RNAP and double the overall transcription rate at the expense of fidelity. Moreover, we monitored single RNAPs coupled to ribosomes and show that coupling increases the pause-free velocity of the polymerase and that a mechanical assisting force is sufficient to explain the majority of the effects of coupling. Also, by cryo-EM, we observed that RNAPs with a terminal mismatch adopt a backtracked conformation, while a coupled ribosome allosterically induces these polymerases toward a catalytically active anti-swiveled state. Finally, we demonstrate that prolonged RNAP pausing is detrimental to cell viability, which could be prevented by polymerase reactivation through a coupled ribosome.

Keywords: RNA polymerase; allostery; coupling; fidelity; force; ribosome; transcription; translation.

Publication types

Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

DNA-Directed RNA Polymerases / genetics
Escherichia coli / genetics
Escherichia coli / metabolism
Escherichia coli Proteins* / genetics
Ribosomes / metabolism
Transcription, Genetic*

Substances

DNA-Directed RNA Polymerases
Escherichia coli Proteins

Abstract

Publication types

MeSH terms

Substances

Grants and funding