The elemental mechanism of transcriptional pausing

Saba, Jason; Chua, Xien Yu; Mishanina, Tatiana V.; Nayak, Dhananjaya; Windgassen, Tricia A.; Mooney, Rachel A.; Landick, Robert

doi:10.7554/elife.40981

Cited by 67 publications

(106 citation statements)

References 70 publications

(176 reference statements)

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“…We believe that this might be the reason behind our observation regarding the limited effect of GreB on the averege backtracking durations. These findings are consistent with (20,51).…”

Section: Average Backtracking Durations Show Little Change In the Presupporting

confidence: 92%

Residence Time Analysis of RNA Polymerase Transcription Dynamics: A Bayesian Sticky HMM Approach

Kilic

Sgouralis

Pressé

2020

Preprint

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The time spent by a single RNA polymerase (RNAP) at specific locations along the DNA, termed “residence time”, reports on the initiation, elongation and termination stages of transcription. At the single molecule level, this information can be obtained from dual ultra-stable optical trapping experiments, revealing a transcriptional elongation of RNAP interspersed with residence times of variable duration. Successfully discriminating between long and short residence times was used by previous approaches to learn about RNAP’s transcription elongation dynamics. Here, we propose an approach based on the Bayesian sticky hidden Markov models that treats all residence times, for an E. Coli RNAP, on an equal footing without a priori discriminating between long and short residence times. In addition, our method has two additional advantages, we provide: full distributions around key point statistics; and directly treat the sequence-dependence of RNAP’s elongation rate.By applying our approach to experimental data, we find: no emergent separation between long and short residence times warranted by the data; force dependent average residence time transcription elongation dynamics; limited effects of GreB on average backtracking durations and counts; and a slight drop in the average residence time as a function of applied force in RNaseA’s presence.STATEMENT OF SIGNIFICANCEMuch of what we know about RNA Polymerase, and its associated transcription factors, relies on successfully discriminating between what are believed to be short and long residence times in the data. This is achieved by applying pause-detection algorithms to trace analysis. Here we propose a new method relying on Bayesian sticky hidden Markov models to interpret time traces provided by dual optical trapping experiments associated with transcription elongation of RNAP. Our method does not discriminate between short and long residence times from the offset in the analysis. It allows for DNA site-dependent transition probabilities of RNAP to neighboring sites (thereby accounting for chemical variability in site to site transitions) and does not demand any time trace pre-processing (such as denoising).

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“…We believe that this might be the reason behind our observation regarding the limited effect of GreB on the averege backtracking durations. These findings are consistent with (20,51).…”

Section: Average Backtracking Durations Show Little Change In the Presupporting

confidence: 92%

Residence Time Analysis of RNA Polymerase Transcription Dynamics: A Bayesian Sticky HMM Approach

Kilic

Sgouralis

Pressé

2020

Preprint

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“…In E. coli, a special pausing signal G-10Y-1G+1 (Y-1 represents the pause site) was identified [5][6][7] , which slows down RNA polymerase (RNAP) near a translation start site allowing coordination of RNAP movement with cotranscriptional translation. The elemental pause could be further stabilized by an RNA hairpin formed in the RNA exit channel of RNAP 8,9 or by RNAP backtracking 10,11 . During backtracking, RNAP moves backward along the DNA and the nascent RNA leading to extrusion of the RNA 3' end into the RNAP secondary channel to induce long pause or transcription arrest 10,12 .…”

Section: Introductionmentioning

confidence: 99%

Nascent RNA sequencing identifies a widespread sigma70-dependent pausing regulated by Gre factors in bacteria

Sun

Yakhnin

Fitzgerald

et al. 2020

Preprint

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Promoter-proximal pausing regulates expression of many eukaryotic genes and serves as checkpoints for assembly of elongation/splicing machinery. Little is known how broadly the pausing is employed in transcriptional regulation in bacteria. We applied NET-seq combined with RNase I footprinting for genome-wide analysis of σ70-dependent transcription pauses in Escherichia coli. Many E. coli genes appear to contain clusters of strong backtracked pauses at 10-20-bp distance from the transcription start site caused by retention of σ70 subunit in RNA polymerase. The pauses in 10-15-bp register of the promoter are dictated by binding of σ70 to canonical -10 element, 6-7 nt spacer and ″YR+1Y″ motif centered at transcription start site all characteristic for strong E. coli promoters. The promoters for the pauses in 16-20-bp register contain an additional -10-like sequence positioned on the same face of the DNA duplex as the original -10 element suggesting that σ70 hopping was responsible for these pauses. Our in vitro analysis reveals that RNA polymerase backtracking and DNA scrunching are involved in these pauses that are relieved by Gre transcript cleavage factors. The genes coding for transcription factors are enriched in these pauses suggesting that σ70 and Gre proteins regulate transcription in response to changing environmental cues.

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“…However, the addition of 2 μM of GreA completely abolished the OZM induced arrest at +7 suggesting that the arrest was predominantly due to backtracking. While the consensus pause is known to be fractionally backtracked, GreA does not speed up the RNAP escape from the consensus pause site (96,98). We concluded that the pause sequence most likely solely provided time for the TEC to backtrack but was not itself a rate limiting step for the recovery from the OZM-induced arrest.…”

Section: Resultsmentioning

confidence: 99%

“…Overall, our data suggested that OZM arrested transcription exclusively by promoting backtracking. While the consensus-pause-like sequence was involved in arresting RNAP in one case, the alleviation of the arrest by GreA suggested that the escape from the pause was not rate limiting (96,98), and that the pause merely provided time for entry into the backtracked state by a fraction of the transcribing RNAP. Noteworthy, a synthetic pyrazine-carboxamide nucleotide analog T-1106 has been recently suggested to inhibit viral RNA-dependent RNA polymerase by promoting backtracking (99).…”

Section: Discussionmentioning

confidence: 99%

Oxazinomycin arrests RNA polymerase at the polythymidine sequences

Prajapati

Rosenqvist

Palmu

et al. 2019

Nucleic Acids Research

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Oxazinomycin is a C-nucleoside antibiotic that is produced by Streptomyces hygroscopicus and closely resembles uridine. Here, we show that the oxazinomycin triphosphate is a good substrate for bacterial and eukaryotic RNA polymerases (RNAPs) and that a single incorporated oxazinomycin is rapidly extended by the next nucleotide. However, the incorporation of several successive oxazinomycins or a single oxazinomycin in a certain sequence context arrested a fraction of the transcribing RNAP. The addition of Gre RNA cleavage factors eliminated the transcriptional arrest at a single oxazinomycin and shortened the nascent RNAs arrested at the polythymidine sequences suggesting that the transcriptional arrest was caused by backtracking of RNAP along the DNA template. We further demonstrate that the ubiquitous C-nucleoside pseudouridine is also a good substrate for RNA polymerases in a triphosphorylated form but does not inhibit transcription of the polythymidine sequences. Our results collectively suggest that oxazinomycin functions as a Trojan horse substrate and its inhibitory effect is attributable to the oxygen atom in the position corresponding to carbon five of the uracil ring.

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The elemental mechanism of transcriptional pausing

Cited by 67 publications

References 70 publications

Residence Time Analysis of RNA Polymerase Transcription Dynamics: A Bayesian Sticky HMM Approach

Residence Time Analysis of RNA Polymerase Transcription Dynamics: A Bayesian Sticky HMM Approach

Nascent RNA sequencing identifies a widespread sigma70-dependent pausing regulated by Gre factors in bacteria

Oxazinomycin arrests RNA polymerase at the polythymidine sequences

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