A1 Refereed original research article in a scientific journal
Active site closure stabilizes the backtracked state of RNA polymerase
Authors: Turtola M, Mäkinen JJ, Belogurov GA
Publisher: Oxford University Press
Publication year: 2018
Journal: Nucleic Acids Research
Volume: 46
Issue: 20
First page : 10870
Last page: 10887
Number of pages: 18
ISSN: 0305-1048
eISSN: 1362-4962
DOI: https://doi.org/10.1093/nar/gky883
Web address : https://academic.oup.com/nar/article/46/20/10870/5107003
Self-archived copy’s web address: https://research.utu.fi/converis/portal/detail/Publication/36657563
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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