Oxazinomycin arrests RNA polymerase at the polythymidine sequences

Prajapati, Ranjit Kumar; Rosenqvist, Petja; Palmu, Kaisa; Mäkinen, Janne; Malinen, Anssi M.; Virta, Pasi; Metsä‐Ketelä, Mikko; Belogurov, Georgiy A.

doi:10.1093/nar/gkz782

Cited by 14 publications

(18 citation statements)

References 101 publications

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“…For the DSI3 RNAP, the K m,app value for nucleotide incorporation on the CPD template was 560 6 110 mM (Fig. 7), significantly higher than previously reported K m values for undamaged templates (54,55). For the G1136M(DSI3) RNAP, this value was decreased to 120 6 15 mM (Fig.…”

Section: Mutations In the Tl Decrease K M For Nucleotide Substrates On Damaged Templatesmentioning

confidence: 62%

Role of the trigger loop in translesion RNA synthesis by bacterial RNA polymerase

Agapov

Ignatov

Turtola

et al. 2020

Journal of Biological Chemistry

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DNA lesions can severely compromise transcription and block RNA synthesis by RNA polymerase (RNAP), leading to subsequent recruitment of DNA repair factors to the stalled transcription complex. Recent structural studies have uncovered molecular interactions of several DNA lesions within the transcription elongation complex. However, little is known about the role of key elements of the RNAP active site in translesion transcription. Here, using recombinantly expressed proteins, in vitro transcription, kinetic analyses, and in vivo cell viability assays, we report that point amino acid substitutions in the trigger loop, a flexible element of the active site involved in nucleotide addition, can stimulate translesion RNA synthesis by Escherichia coli RNAP without altering the fidelity of nucleotide incorporation. We show that these substitutions also decrease transcriptional pausing and strongly affect the nucleotide addition cycle of RNAP by increasing the rate of nucleotide addition but also decreasing the rate of translocation. The secondary channel factors DksA and GreA modulated translesion transcription by RNAP, depending on changes in the trigger loop structure. We observed that although the mutant RNAPs stimulate translesion synthesis, their expression is toxic in vivo, especially under stress conditions. We conclude that the efficiency of translesion transcription can be significantly modulated by mutations affecting the conformational dynamics of the active site of RNAP, with potential effects on cellular stress responses and survival.

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Section: Mutations In the Tl Decrease K M For Nucleotide Substrates On Damaged Templatesmentioning

confidence: 62%

Role of the trigger loop in translesion RNA synthesis by bacterial RNA polymerase

Agapov

Ignatov

Turtola

et al. 2020

Journal of Biological Chemistry

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“…S4B ; Methods M9 ). Their precise values are expected to vary for different nucleotides (9, 36, 37). We found that the values of k el and P total estimated by the fits are well captured by Eqs.…”

Section: Resultsmentioning

confidence: 99%

Accounting for RNA polymerase heterogeneity reveals state switching and two distinct long-lived backtrack states escaping through cleavage

Janissen

Eslami-Mossallam

Artsimovitch

et al. 2019

Preprint

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Pausing by RNA polymerase (RNAp) facilitates the recruitment of regulatory factors, RNA folding, and other related processes. While backtracking and intra-structural isomerization have been proposed to trigger pausing, the mechanisms of pause formation and recovery remain debated. Using high-throughput magnetic tweezers with single-molecule sensitivity, we have examined the full temporal spectrum of Escherichia coli RNAp transcription dynamics. Together with probabilistic dwell-time analysis and modelling, this has identified three distinct states that compete with elongation: a short-lived elemental pause and two longlived backtracked pause states. We demonstrate that recovery from backtracking is not governed by diffusional Brownian motion, but rather by intrinsic RNA cleavage, whereby RNAp conformational changes hinder cleavage in long-lived pause states. We further show that state switching underlies stochastic alterations in the frequency of short pauses. Based on these findings, we propose a consensus model of intrinsic pausing that unifies all key findings while resolving earlier contradictions.

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“…β'Q929M, Supplementary Fig. 2) could not be adequately fit by the previously employed reversible inactivation model 28,45,56 .…”

Section: Scenariomentioning

confidence: 91%

“…Overall, fits to Scheme 1 were performed largely as described in Prajapati et al 45 except that here we used a non-equilibrium heterogeneity model (see below) to describe the kinetic heterogeneity in the TEC preparations instead of a reversible inactivation model (see below)…”

Section: Supplementarymentioning

confidence: 99%

The mechanism of the nucleo-sugar selection by multi-subunit RNA polymerases

Mäkinen

Shin

Vieras

et al. 2020

Preprint

Self Cite

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AbstractRNA polymerases (RNAPs) synthesize RNA from NTPs, whereas DNA polymerases synthesize DNA from 2’dNTPs. DNA polymerases select against NTPs by using steric gates to exclude the 2’ OH, but RNAPs have to employ alternative selection strategies. In single-subunit RNAPs, a conserved Tyr residue discriminates against 2’dNTPs, whereas selectivity mechanisms of multi-subunit RNAPs remain hitherto unknown. Here we show that a conserved Arg residue uses a two-pronged strategy to select against 2’dNTPs in multi-subunit RNAPs. The conserved Arg interacts with the 2’OH group to promote NTP binding, but selectively inhibits incorporation of 2’dNTPs by interacting with their 3’OH group to favor the catalytically-inert 2’-endo conformation of the deoxyribose moiety. This deformative action is an elegant example of an active selection against a substrate that is a substructure of the correct substrate. Our findings provide important insights into the evolutionary origins of biopolymers and the design of selective inhibitors of viral RNAPs.

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Oxazinomycin arrests RNA polymerase at the polythymidine sequences

Cited by 14 publications

References 101 publications

Role of the trigger loop in translesion RNA synthesis by bacterial RNA polymerase

Role of the trigger loop in translesion RNA synthesis by bacterial RNA polymerase

Accounting for RNA polymerase heterogeneity reveals state switching and two distinct long-lived backtrack states escaping through cleavage

The mechanism of the nucleo-sugar selection by multi-subunit RNA polymerases

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