The elemental mechanism of transcriptional pausing

Transcriptional pausing underlies regulation of cellular RNA biogenesis. A consensus pause sequence that acts on RNA polymerases (RNAPs) from bacteria to mammals halts RNAP in an elemental paused state from which longer-lived pauses can arise. Although the structural foundations of pauses prolonged by backtracking or nascent RNA hairpins are recognized, the fundamental mechanism of the elemental pause is less well-defined. Here we report a mechanistic dissection that establishes the elemental pause signal (i) is multipartite; (ii) causes a modest conformational shift that puts γ-proteobacterial RNAP in an off-pathway state in which template base loading but not RNA translocation is inhibited; and (iii) allows RNAP to enter pretranslocated and one-base-pair backtracked states easily even though the half-translocated state observed in paused cryo-EM structures rate-limits pause escape. Our findings provide a mechanistic basis for the elemental pause and a framework to understand how pausing is modulated by sequence, cellular conditions, and regulators.

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The elemental mechanism of transcriptional pausing

10.1101/422220 ◽

2018 ◽

Cited By ~ 1

Author(s):

Jason Saba ◽

Xien Chua ◽

Tatiana V. Mishanina ◽

Dhananjaya Nayak ◽

Tricia A. Windgassen ◽

...

Keyword(s):

Base Pair ◽

Rna Polymerases ◽

Rna Hairpins ◽

Rna Biogenesis ◽

Transcriptional Pausing ◽

Nascent Rna ◽

Mechanistic Basis ◽

Fundamental Mechanism

ABSTRACTTranscriptional pausing underpins regulation of cellular RNA biogenesis. A consensus pause sequence that acts on RNA polymerases (RNAPs) from bacteria to mammals halts RNAP in an elemental paused state from which longer-lived pauses can arise. Although the structural foundations of pauses prolonged by backtracking or nascent RNA hairpins are recognized, the fundamental mechanism of the elemental pause is less well-defined. Here we report a mechanistic dissection that establishes the elemental pause signal (i) is multipartite; (ii) causes a modest conformational shift that puts RNAP in an off-pathway state in which template base loading but not RNA translocation is inhibited; and (iii) can easily enter pretranslocated and one-base-pair backtracked states despite principally occupying the half-translocated state observed in cryo-EM structures of paused RNAPs. Our findings provide a mechanistic basis for the elemental pause and a framework to understand how pausing is modulated by sequence, cellular conditions, and regulators.

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Faculty Opinions recommendation of Global analysis of nascent RNA reveals transcriptional pausing in terminal exons.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.9206959.9800057 ◽

2011 ◽

Author(s):

Andrea Barta

Keyword(s):

Global Analysis ◽

Transcriptional Pausing ◽

Nascent Rna

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Transcriptional Pausing in Vivo: A Nascent RNA Hairpin Restricts Lateral Movements of RNA Polymerase in Both Forward and Reverse Directions

Journal of Molecular Biology ◽

10.1016/j.jmb.2005.05.052 ◽

2005 ◽

Vol 351 (1) ◽

pp. 39-51 ◽

Cited By ~ 18

Author(s):

Francine Toulmé ◽

Christine Mosrin-Huaman ◽

Irina Artsimovitch ◽

A. Rachid Rahmouni

Keyword(s):

Rna Polymerase ◽

Transcriptional Pausing ◽

Nascent Rna ◽

Rna Hairpin

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The regulatory roles and mechanism of transcriptional pausing

Biochemical Society Transactions ◽

10.1042/bst0341062 ◽

2006 ◽

Vol 34 (6) ◽

pp. 1062-1066 ◽

Cited By ~ 170

Author(s):

R. Landick

Keyword(s):

Single Molecule ◽

Life Forms ◽

Eukaryotic Transcription ◽

Regulatory Interactions ◽

Enzymatic Mechanism ◽

Cellular Life ◽

Efficient Gene ◽

Transcriptional Pausing ◽

Elongation Phase ◽

Fundamental Mechanism

The multisubunit RNAPs (RNA polymerases) found in all cellular life forms are remarkably conserved in fundamental structure, in mechanism and in their susceptibility to sequence-dependent pausing during transcription of DNA in the absence of elongation regulators. Recent studies of both prokaryotic and eukaryotic transcription have yielded an increasing appreciation of the extent to which gene regulation is accomplished during the elongation phase of transcription. Transcriptional pausing is a fundamental enzymatic mechanism that underlies many of these regulatory schemes. In some cases, pausing functions by halting RNAP for times or at positions required for regulatory interactions. In other cases, pauses function by making RNAP susceptible to premature termination of transcription unless the enzyme is modified by elongation regulators that programme efficient gene expression. Pausing appears to occur by a two-tiered mechanism in which an initial rearrangement of the enzyme's active site interrupts active elongation and puts RNAP in an elemental pause state from which additional rearrangements or regulator interactions can create long-lived pauses. Recent findings from biochemical and single-molecule transcription experiments, coupled with the invaluable availability of RNAP crystal structures, have produced attractive hypotheses to explain the fundamental mechanism of pausing.

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Control of noncoding RNA production and histone levels by a 5′ tRNA fragment

10.1101/498949 ◽

2018 ◽

Cited By ~ 1

Author(s):

Ana Boskovic ◽

Xin Yang Bing ◽

Ebru Kaymak ◽

Oliver J Rando

Keyword(s):

Transcriptional Repression ◽

Noncoding Rna ◽

Rna Binding ◽

Rna Binding Proteins ◽

Noncoding Rnas ◽

Cajal Body ◽

Mrna Levels ◽

Histone 3 ◽

Rna Biogenesis ◽

Mechanistic Basis

Small RNAs derived from mature tRNAs, referred to as tRNA fragments or "tRFs", are an emerging class of regulatory RNAs with poorly understood functions in cellular regulation. We recently identified a role for one specific tRF - 5′ tRF-Gly-GCC, or tRF-GG - in repression of genes associated with the endogenous retroelement MERVL, but the mechanistic basis for this regulation was unknown. Here, we show that tRF-GG plays a role in production of a wide variety of noncoding RNAs normally synthesized in Cajal bodies. Among these noncoding RNAs, tRF-GG regulation of the U7 snRNA modulates heterochromatin-mediated transcriptional repression of MERVL elements by supporting an adequate supply of histone proteins. Importantly, the effects of inhibiting tRF-GG on histone mRNA levels, activity of a histone 3′ UTR reporter, and ultimately on MERVL regulation could all be suppressed by the U7 RNA. We show that the related RNA-binding proteins hnRNPF and H bind directly to tRF-GG, and are required for Cajal body biogenesis. Together, our data reveal a conserved mechanism for 5′ tRNA fragment control of noncoding RNA biogenesis and, consequently, in global chromatin organization.

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