A1 Refereed original research article in a scientific journal

Active site closure stabilizes the backtracked state of RNA polymerase




AuthorsTurtola M, Mäkinen JJ, Belogurov GA

PublisherOxford University Press

Publication year2018

JournalNucleic Acids Research

Volume46

Issue20

First page 10870

Last page10887

Number of pages18

ISSN0305-1048

eISSN1362-4962

DOIhttps://doi.org/10.1093/nar/gky883

Web address https://academic.oup.com/nar/article/46/20/10870/5107003

Self-archived copy’s web addresshttps://research.utu.fi/converis/portal/detail/Publication/36657563


Abstract

All cellular RNA polymerases (RNAP) occasionally backtrack along the template DNA as part of transcriptional proofreading and regulation. Here, we studied the mechanism of RNAP backtracking by one nucleotide using two complementary approaches that allowed us to precisely measure the occupancy and lifetime of the backtracked state. Our data show that the stability of the backtracked state is critically dependent on the closure of the RNAP active site by a mobile domain, the trigger loop (TL). The lifetime and occupancy of the backtracked state measurably decreased by substitutions of the TL residues that interact with the nucleoside triphosphate (NTP) substrate, whereas amino acid substitutions that stabilized the closed active site increased the lifetime and occupancy. These results suggest that the same conformer of the TL closes the active site during catalysis of nucleotide incorporation into the nascent RNA and backtracking by one nucleotide. In support of this hypothesis, we construct a model of the 1-nt backtracked complex with the closed active site and the backtracked nucleotide in the entry pore area known as the E-site. We further propose that 1-nt backtracking mimics the reversal of the NTP substrate loading into the RNAP active site during on-pathway elongation.


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