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An NTP-driven mechanism for the nucleotide addition cycle of Escherichia coli RNA polymerase during transcription

The elementary steps of transcription as catalyzed by E. coli RNA polymerase during one and two rounds of the nucleotide addition cycle (NAC) were resolved in rapid kinetic studies. Modelling of stopped-flow kinetic data of pyrophosphate release in a coupled enzyme assay during one round of the NAC...

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Autores principales: Johnson, Ronald S., Strausbauch, Mark, McCloud, Christopher
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9595533/
https://www.ncbi.nlm.nih.gov/pubmed/36282801
http://dx.doi.org/10.1371/journal.pone.0273746
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author Johnson, Ronald S.
Strausbauch, Mark
McCloud, Christopher
author_facet Johnson, Ronald S.
Strausbauch, Mark
McCloud, Christopher
author_sort Johnson, Ronald S.
collection PubMed
description The elementary steps of transcription as catalyzed by E. coli RNA polymerase during one and two rounds of the nucleotide addition cycle (NAC) were resolved in rapid kinetic studies. Modelling of stopped-flow kinetic data of pyrophosphate release in a coupled enzyme assay during one round of the NAC indicates that the rate of pyrophosphate release is significantly less than that for nucleotide incorporation. Upon modelling of the stopped-flow kinetic data for pyrophosphate release during two rounds of the NAC, it was observed that the presence of the next nucleotide for incorporation increases the rate of release of the first pyrophosphate equivalent; incorrect nucleotides for incorporation had no effect on the rate of pyrophosphate release. Although the next nucleotide for incorporation increases the rate of pyrophosphate release, it is still significantly less than the rate of incorporation of the first nucleotide. The results from the stopped-flow kinetic studies were confirmed by using quench-flow followed by thin-layer chromatography (QF-TLC) with only the first nucleotide for incorporation labeled on the gamma phosphate with (32)P to monitor pyrophosphate release. Collectively, the results are consistent with an NTP-driven model for the NAC in which the binding of the next cognate nucleotide for incorporation causes a synergistic conformational change in the enzyme that triggers the more rapid release of pyrophosphate, translocation of the enzyme along the DNA template strand and nucleotide incorporation.
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spelling pubmed-95955332022-10-26 An NTP-driven mechanism for the nucleotide addition cycle of Escherichia coli RNA polymerase during transcription Johnson, Ronald S. Strausbauch, Mark McCloud, Christopher PLoS One Research Article The elementary steps of transcription as catalyzed by E. coli RNA polymerase during one and two rounds of the nucleotide addition cycle (NAC) were resolved in rapid kinetic studies. Modelling of stopped-flow kinetic data of pyrophosphate release in a coupled enzyme assay during one round of the NAC indicates that the rate of pyrophosphate release is significantly less than that for nucleotide incorporation. Upon modelling of the stopped-flow kinetic data for pyrophosphate release during two rounds of the NAC, it was observed that the presence of the next nucleotide for incorporation increases the rate of release of the first pyrophosphate equivalent; incorrect nucleotides for incorporation had no effect on the rate of pyrophosphate release. Although the next nucleotide for incorporation increases the rate of pyrophosphate release, it is still significantly less than the rate of incorporation of the first nucleotide. The results from the stopped-flow kinetic studies were confirmed by using quench-flow followed by thin-layer chromatography (QF-TLC) with only the first nucleotide for incorporation labeled on the gamma phosphate with (32)P to monitor pyrophosphate release. Collectively, the results are consistent with an NTP-driven model for the NAC in which the binding of the next cognate nucleotide for incorporation causes a synergistic conformational change in the enzyme that triggers the more rapid release of pyrophosphate, translocation of the enzyme along the DNA template strand and nucleotide incorporation. Public Library of Science 2022-10-25 /pmc/articles/PMC9595533/ /pubmed/36282801 http://dx.doi.org/10.1371/journal.pone.0273746 Text en © 2022 Johnson et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Johnson, Ronald S.
Strausbauch, Mark
McCloud, Christopher
An NTP-driven mechanism for the nucleotide addition cycle of Escherichia coli RNA polymerase during transcription
title An NTP-driven mechanism for the nucleotide addition cycle of Escherichia coli RNA polymerase during transcription
title_full An NTP-driven mechanism for the nucleotide addition cycle of Escherichia coli RNA polymerase during transcription
title_fullStr An NTP-driven mechanism for the nucleotide addition cycle of Escherichia coli RNA polymerase during transcription
title_full_unstemmed An NTP-driven mechanism for the nucleotide addition cycle of Escherichia coli RNA polymerase during transcription
title_short An NTP-driven mechanism for the nucleotide addition cycle of Escherichia coli RNA polymerase during transcription
title_sort ntp-driven mechanism for the nucleotide addition cycle of escherichia coli rna polymerase during transcription
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9595533/
https://www.ncbi.nlm.nih.gov/pubmed/36282801
http://dx.doi.org/10.1371/journal.pone.0273746
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