T7 RNA polymerase studied by force measurements varying cofactor concentration.

Biophysical Journal
Philippe ThomenFrançois Heslot

Abstract

RNA polymerases carry out the synthesis of an RNA copy from a DNA template. They move along DNA, incorporate nucleotide triphosphate (NTP) at the end of the growing RNA chain, and consume chemical energy. In a single-molecule assay using the T7 RNA polymerase, we study how a mechanical force opposing the forward motion of the enzyme along DNA affects the translocation rate. We also study the influence of nucleotide and magnesium concentration on this process. The experiment shows that the opposing mechanical force is a competitive inhibitor of nucleotide binding. Also, the single-molecule data suggest that magnesium ions are involved in a step that does not depend on the external load force. These kinetic results associated with known biochemical and mutagenic data, along with the static information obtained from crystallographic structures, shape a very coherent view of the catalytic cycle of the enzyme: translocation does not take place upon NTP binding nor upon NTP cleavage, but rather occurs after PPi release and before the next nucleotide binding event. Furthermore, the energetic bias associated with the forward motion of the enzyme is close to kT and represents only a small fraction of the free energy of nucleotide incorp...Continue Reading

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Citations

Sep 24, 2013·Chemical Reviews·Jens Michaelis, Barbara Treutlein
May 23, 2012·Proceedings of the National Academy of Sciences of the United States of America·Bradley ZamftCarlos Bustamante
Jul 23, 2013·PLoS Computational Biology·Joshua I GlaserKonrad P Kording
Jan 30, 2014·ELife·Jordan Monnet, Terence R Strick
Feb 16, 2011·Biophysical Journal·Gary M SkinnerKoen Visscher
Mar 28, 2013·Bacteriophage·Philip Serwer, Wen Jiang
Jul 28, 2016·Nature Communications·Katjana TantaleEdouard Bertrand
Jan 22, 2014·Chemical Reviews·Iddo HellerGijs J L Wuite
Oct 9, 2009·Current Opinion in Structural Biology·Thomas A Steitz
Feb 18, 2006·Journal of Molecular Biology·Lisa M JungbauerSilvia Cavagnero
Feb 15, 2020·PLoS Computational Biology·Jordan DouglasAlexei J Drummond
Oct 3, 2019·Biotechnology and Applied Biochemistry·Xumeng Ge, Jianfeng Xu
Mar 25, 2017·Nature Chemistry·Shunsuke TagamiPhilipp Holliger
Dec 20, 2020·Biophysical Journal·Keith J MickolajczykTarun M Kapoor
Aug 17, 2021·Journal of the American Chemical Society·Ze YuMichael Famulok

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