Structural and mechanistic basis of σ-dependent transcriptional pausing (original) (raw)
Related papers
Ubiquitous Transcriptional Pausing Is Independent of RNA Polymerase Backtracking
Cell, 2003
that interact with the transcription elongation complex 1 Department of Biological Sciences (TEC) (e.g., N and Q; Richardson and Greenblatt, 1996; Stanford University Roberts et al., 1998; Weisberg and Gottesman, 1999; Stanford, California 94305 and references therein). Finally, frequent pausing is ob-2 Department of Applied Physics served during the transcription of genomic DNA (Kassa-Stanford University vetis and Chamberlin, 1981; Matsuzaki et al., 1994; Adel-Stanford, California 94305 man et al., 2002). For bacterial RNAP, such pauses are 3 Department of Bacteriology thought to limit the overall rate of transcription, thereby University of Wisconsin synchronizing transcription with translation (translating Madison, Wisconsin 53706 ribosomes release paused RNAPs), and allowing Rho-4 Department of Biochemistry catalyzed termination of transcription should translation Brandeis University fail (Landick et al., 1985; Richardson and Greenblatt, Waltham, Massachusetts 02454 1996). Both bulk (Kassavetis and Chamberlin, 1981; Theissen et al., 1990; Matsuzaki et al., 1994; Wang et al., 1995) Summary and single-molecule (Davenport et al., 2000; Adelman et al., 2002; Forde et al., 2002) transcription experiments RNA polymerase (RNAP) transcribes DNA discontinu-
Proceedings of the National Academy of Sciences, 1997
RNA secondary structures (hairpins) that form as the nascent RNA emerges from RNA polymerase are important components of many signals that regulate transcription, including some pause sites, all -independent terminators, and some antiterminators. At the his leader pause site, a 5-bp-stem, 8-nt-loop pause RNA hairpin forms 11 nt from the RNA 3 end and stabilizes a transcription complex conformation slow to react with NTP substrate. This stabilization appears to depend at least in part on an interaction with RNA polymerase. We tested for RNA hairpin interaction with the paused polymerase by crosslinking 5-iodoUMP positioned specifically in the hairpin loop. In the paused conformation, strong and unusual crosslinking of the pause hairpin to 904-950 replaced crosslinking to  and to other parts of  that occurred in nonpaused complexes prior to hairpin formation. These changes in nascent RNA interactions may inhibit reactive alignment of the RNA 3 end in the paused complex and be related to events at -independent terminators.
Proceedings of the National Academy of Sciences of the United States of America, 1997
RNA secondary structures (hairpins) that form as the nascent RNA emerges from RNA polymerase are important components of many signals that regulate transcription, including some pause sites, all ρ-independent terminators, and some antiterminators. At the his leader pause site, a 5-bp-stem, 8-nt-loop pause RNA hairpin forms 11 nt from the RNA 3′ end and stabilizes a transcription complex conformation slow to react with NTP substrate. This stabilization appears to depend at least in part on an interaction with RNA polymerase. We tested for RNA hairpin interaction with the paused polymerase by crosslinking 5-iodoUMP positioned specifically in the hairpin loop. In the paused conformation, strong and unusual crosslinking of the pause hairpin to β904–950 replaced crosslinking to β′ and to other parts of β that occurred in nonpaused complexes prior to hairpin formation. These changes in nascent RNA interactions may inhibit reactive alignment of the RNA 3′ end in the paused complex and be related to events at ρ-independent terminators.
Different types of pausing modes during transcription initiation
Transcription, 2017
In many cases, initiation is rate limiting to transcription. This due in part to the multiple cycles of abortive transcription that delay promoter escape and the transition from initiation to elongation. Pausing of transcription in initiation can further delay promoter escape. The previously hypothesized pausing in initiation was confirmed by two recent studies from Duchi et al. 1 and from Lerner, Chung et al. 2 In both studies, pausing is attributed to a lack of forward translocation of the nascent transcript during initiation. However, the two works report on different pausing mechanisms. Duchi et al. report on pausing that occurs during initiation predominantly on-pathway of transcript synthesis. Lerner, Chung et al. report on pausing during initiation as a result of RNAP backtracking, which is off-pathway to transcript synthesis. Here, we discuss these studies, together with additional experimental results from single-molecule FRET focusing on a specific distance within the transcription bubble. We show that the results of these studies are complementary to each other and are consistent with a model involving two types of pauses in initiation: a short-lived pause that occurs in the translocation of a 6-mer nascent transcript and a long-lived pause that occurs as a result of 1-2 nucleotide backtracking of a 7-mer transcript.
Evolution of promoter-proximal pausing enabled a new layer of transcription control
bioRxiv (Cold Spring Harbor Laboratory), 2023
Promoter-proximal pausing of RNA polymerase II (Pol II) is a key regulatory step during transcription. To understand the evolution and function of pausing, we analyzed transcription in 20 organisms across the tree of life. Unicellular eukaryotes have a slow acceleration of Pol II near transcription start sites that matured into a longer and more focused pause in metazoans. Increased pause residence time coincides with the evolution of new subunits in the NELF and 7SK complexes. In mammals, depletion of NELF reverts a focal pause to a proto-paused-like state driven in part by DNA sequence. Loss of this focal pause compromises transcriptional activation for a set of heat shock genes. Overall, we discovered how pausing evolved and increased regulatory complexity in metazoans.
Nature communications, 2018
Transcription by RNA polymerase (RNAP) is interspersed with sequence-dependent pausing. The processes through which paused states are accessed and stabilized occur at spatiotemporal scales beyond the resolution of previous methods, and are poorly understood. Here, we combine high-resolution optical trapping with improved data analysis methods to investigate the formation of paused states at enhanced temporal resolution. We find that pause sites reduce the forward transcription rate of nearly all RNAP molecules, rather than just affecting the subset of molecules that enter long-lived pauses. We propose that the reduced rates at pause sites allow time for the elongation complex to undergo conformational changes required to enter long-lived pauses. We also find that backtracking occurs stepwise, with states backtracked by at most one base pair forming quickly, and further backtracking occurring slowly. Finally, we find that nascent RNA structures act as modulators that either enhance o...
2007
Although establishing the detailed kinetic mechanism of nucleotide addition and pausing is well beyond the scope of this study, we wished to test minimal pause mechanisms to determine whether our mechanistic conclusions were reasonable. We emphasize that these mechanisms cannot fully account for the observed NTP-dependence of hairpin-stabilized (e. g. his leader) pausing, but can account for the basic characteristics of pausing. In particular, the mechanisms do not include steps to account for NTP-stimulation of translocation, the existence of which is supported by both ensemble and single-molecule experiments . With this caveat noted, examining these mechanisms can help evaluate basic properties of the his PTC and the predictions of structure/function experiments. We first tested the simplest possible pause mechanism (mechanism 1), in which a single off-line state forms by isomerization of the pretranslocated EC (e.g., by fraying of the RNA 3´ nt, rearrangement of active-site residues, trapping of the TL in an inhibitory conformation, or some combination of these events) and slow escape of the PTC (U p pre ) back to the pretranslocated EC (U pre ). In this mechanism, the elemental pause and hairpinstabilized states are lumped together on the assumption that hairpin formation is rapid enough to make them kinetically indistinguishable.
Journal of Biological Chemistry
We have identified minimal nucleic acid scaffolds capable of reconstituting hairpin-stabilized paused transcription complexes when incubated with RNAP either directly or in a limited step reconstitution assay. Direct reconstitution was achieved using a 29-nucleotide (nt) RNA whose 3-proximal 9 -10 nt pair to template DNA within an 11-nt noncomplementary bubble of a 39-bp duplex DNA; the 5-proximal 18 nt of RNA forms the his pause RNA hairpin. Limited-step reconstitution was achieved on the same DNAs using a 27-nt RNA that can be 3-labeled during reconstitution and then extended 2 nt past the pause site to assay transcriptional pausing. Paused complexes formed by either method recapitulated key features of a promoter-initiated, hairpin-stabilized paused complex, including a slow rate of pause escape, resistance to transcript cleavage and pyrophosphorolysis, and enhancement of pausing by the elongation factor NusA. These findings establish that RNA upstream from the pause hairpin and pyrophosphate are not essential for pausing and for NusA action. Reconstitution of the his paused transcription complex provides a valuable tool for future studies of protein-nucleic interactions involved in transcriptional pausing.
Inhibition of a transcriptional pause by RNA anchoring to RNA polymerase
Molecular cell, 2008
We describe a mechanism by which nascent RNA inhibits transcriptional pausing. PutL RNA of bacteriophage HK022 suppresses transcription termination at downstream terminators and pausing within a nearby U-rich sequence. In vitro transcription and footprinting assays reveal that this pausing results from backtracking of RNA polymerase and that binding of nascent putL RNA to polymerase limits backtracking by restricting re-entry of the transcript into the RNA exit channel. The restriction is local and relaxes as the transcript elongates. Our results suggest that putL RNA binds to the surface of polymerase close to the RNA exit channel, a region that includes amino acid residues important for antitermination. Although binding is essential for antipausing and antitermination, these two activities of put differ: antipausing is limited to the immediate vicinity of the putL site, but antitermination is not. We propose that RNA anchoring to the elongation complex is a widespread mechanism of...
Sequence-Resolved Detection of Pausing by Single RNA Polymerase Molecules
Cell, 2006
Transcriptional pausing by RNA polymerase (RNAP) plays an important role in the regulation of gene expression. Defined, sequence-specific pause sites have been identified biochemically. Single-molecule studies have also shown that bacterial RNAP pauses frequently during transcriptional elongation, but the relationship of these ''ubiquitous'' pauses to the underlying DNA sequence has been uncertain. We employed an ultrastable optical-trapping assay to follow the motion of individual molecules of RNAP transcribing templates engineered with repeated sequences carrying imbedded, sequence-specific pause sites of known regulatory function. Both the known and ubiquitous pauses appeared at reproducible locations, identified with base-pair accuracy. Ubiquitous pauses were associated with DNA sequences that show similarities to regulatory pause sequences. Data obtained for the lifetimes and efficiencies of pauses support a model where the transition to pausing branches off of the normal elongation pathway and is mediated by a common elemental state, which corresponds to the ubiquitous pause.