Interactions between RNA polymerase and the “core recognition element” counteract pausing

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During transcription initiation, RNA polymerase (RNAP) holoenzyme unwinds ∼13 bp of promoter DNA, forming an RNAP-promoter open complex (RPo) containing a single-stranded transcription bubble, and selects a template-strand nucleotide to serve as the transcription start site (TSS). In RPo, RNAP core enzyme makes sequence-specific protein-DNA interactions with the downstream part of the nontemplate strand of the transcription bubble ("core recognition element," CRE). Here, we investigated whether sequence-specific RNAP-CRE interactions affect TSS selection. To do this, we used two next-generation sequencing-based approaches to compare the TSS profile of WT RNAP to that of an RNAP derivative defective in sequence-specific RNAP-CRE interactions. First, using massively systematic transcript end readout, MASTER, we assessed effects of RNAP-CRE interactions on TSS selection in vitro and in vivo for a library of 4 7 (∼16,000) consensus promoters containing different TSS region sequences, and we observed that the TSS profile of the RNAP derivative defective in RNAP-CRE interactions differed from that of WT RNAP, in a manner that correlated with the presence of consensus CRE sequences in the TSS region. Second, using 5′ merodiploid nativeelongating-transcript sequencing, 5′ mNET-seq, we assessed effects of RNAP-CRE interactions at natural promoters in Escherichia coli, and we identified 39 promoters at which RNAP-CRE interactions determine TSS selection. Our findings establish RNAP-CRE interactions are a functional determinant of TSS selection. We propose that RNAP-CRE interactions modulate the position of the downstream end of the transcription bubble in RPo, and thereby modulate TSS selection, which involves transcription bubble expansion or transcription bubble contraction (scrunching or antiscrunching).RNA polymerase | transcription start site selection | promoter | transcription bubble | transcription initiation T ranscription initiation consists of a number of biochemical steps leading to formation of a phosphodiester bond between a nucleoside triphosphate (NTP) bound in the RNA polymerase (RNAP) active-center initiating NTP binding site (i site) and an NTP bound in the RNAP active-center extending NTP binding site (i+1 site) (1-3). For bacterial RNAP, promoter-specific initiation requires the RNAP core enzyme (subunit composition α 2 ββ'ω) to associate with a σ factor forming the RNAP holoenzyme (subunit composition α 2 ββ'ωσ). The σ factor contains determinants for sequence-specific protein-DNA interactions with four core promoter elements: the −35 element, the extended −10 element, the −10 element, and the discriminator element (4).During transcription initiation, RNAP holoenzyme unwinds promoter DNA to form an RNAP-promoter open complex (RPo) containing an unwound, single-stranded "transcription bubble." The process of promoter unwinding begins within the promoter −10 element and propagates downstream, enabling single-stranded nucleotides at the downstream end of the transcription bubble template strand to occup...

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection

Vvedenskaya

Vahedian-Movahed

Zhang

et al. 2016

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“…The discriminator region between the −10 hexamer and the transcription start site (TSS; +1) has recently emerged as an important modulator of the open complex properties (11)(12)(13)(14)(15)(16)(17)(18). In a stable open complex formed by Thermus thermophilus RNAP, the −6 and −5 bases of the discriminator NT strand (NT DISC ) make crucial contacts with σ1.2, and βArg371 (E. coli numbering) interacts with the adjacent −4 and −3 bases (14).…”

Section: Resultsmentioning

confidence: 99%

RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes

NandyMazumdar

Nedialkov

Svetlov

et al. 2016

Upon RNA polymerase (RNAP) binding to a promoter, the σ factor initiates DNA strand separation and captures the melted nontemplate DNA, whereas the core enzyme establishes interactions with the duplex DNA in front of the active site that stabilize initiation complexes and persist throughout elongation. Among many core RNAP elements that participate in these interactions, the β′ clamp domain plays the most prominent role. In this work, we investigate the role of the β gate loop, a conserved and essential structural element that lies across the DNA channel from the clamp, in transcription regulation. The gate loop was proposed to control DNA loading during initiation and to interact with NusG-like proteins to lock RNAP in a closed, processive state during elongation. We show that the removal of the gate loop has large effects on promoter complexes, trapping an unstable intermediate in which the RNAP contacts with the nontemplate strand discriminator region and the downstream duplex DNA are not yet fully established. We find that although RNAP lacking the gate loop displays moderate defects in pausing, transcript cleavage, and termination, it is fully responsive to the transcription elongation factor NusG. Together with the structural data, our results support a model in which the gate loop, acting in concert with initiation or elongation factors, guides the nontemplate DNA in transcription complexes, thereby modulating their regulatory properties.D uring each round of transcription, RNA polymerase (RNAP) establishes, maintains, and finally releases contacts with the DNA template and the RNA. These interactions mediate highly selective initiation, processive elongation, and precise termination and can be tuned to enable intricate regulation during the transcription cycle. RNAP resembles a crab claw in which the two pincers composed of the β′ and β subunits form an active site cleft that accommodates the nucleic acid chains (Fig. 1). The mobile β′ clamp domain that forms one of the pincers stands out as a central regulatory feature (1,2). The open clamp likely allows the loading of the promoter DNA during initiation and the release of the template and the nascent RNA during termination. The clamp is thought to close to form an active initiation complex and to remain closed during elongation but may open partially at a hairpindependent pause site (3).The clamp movements may be linked to those of the β lobe domain, which forms a part of the second pincer. The β gate loop (GL), which lies across the RNAP cleft from the tip of the clamp (Fig. 1), has been identified as an element that restricts the entry of the duplex promoter DNA into the narrow active site cleft (4), allowing only a single strand of DNA to pass through. This model posited that the DNA stands must separate outside the cleft before entry into RNAP, whereas footprinting studies demonstrated that the promoter DNA enters the active site cleft before it is opened (5). This controversy was a subject of an intense debate until a recent study revealed that the...

“…S6A) (26,27). Whereas X. oryzae RNAP responded to this type of pausing signal, p7 not only did not suppress pausing on such a site but actually slightly stimulated it (Fig.…”

mentioning

confidence: 87%

Distinct pathways of RNA polymerase regulation by a phage-encoded factor

Esyunina

Klimuk

Severinov

et al. 2015

Transcription antitermination is a common strategy of gene expression regulation, but only a few transcription antitermination factors have been studied in detail. Here, we dissect the transcription antitermination mechanism of Xanthomonas oryzae virus Xp10 protein p7, which binds host RNA polymerase (RNAP) and regulates both transcription initiation and termination. We show that p7 suppresses intrinsic termination by decreasing RNAP pausing and increasing the transcription complex stability, in cooperation with host-encoded factor NusA. Uniquely, the antitermination activity of p7 depends on the ω subunit of the RNAP core and is modulated by ppGpp. In contrast, the inhibition of transcription initiation by p7 does not require ω but depends on other RNAP sites. Our results suggest that p7, a bifunctional transcription factor, uses distinct mechanisms to control different steps of transcription. We propose that regulatory functions of the ω subunit revealed by our analysis may extend to its homologs in eukaryotic RNAPs.RNA polymerase | transcription antitermination | transcription pausing | omega subunit | NusA T ranscription promoters and terminators are the main "punctuation marks" that control the expression of individual genes in the genome. In bacteria, transcription termination is an essential regulatory step that determines the relative levels of expression of promoter-proximal and distal genes within operons. Depending on the factors involved, transcription termination in bacteria can be classified as intrinsic (depends on RNAencoded signals) or factor-dependent (requires additional protein factors such as Rho helicase). Intrinsic terminators consist of a G/C-rich hairpin formed in the nascent RNA followed by a downstream oligo(U) tract. During intrinsic termination, RNA polymerase (RNAP) pauses after synthesizing an oligo(U) sequence, accompanied by hairpin formation, leading to subsequent dissociation of the transcription elongation complex (TEC) (1, 2). Despite the relatively simple overall scenario, the exact mechanisms of pausing and hairpin-induced TEC destabilization remain an issue of debate (1, 3).Transcription antitermination is a widespread phenomenon involved in regulation of bacterial and phage operons (2). Transcription antitermination is often mediated by specialized protein factors that target host RNAP and can suppress termination at multiple sites in the operon. The well-studied examples of phage antiterminators include proteins N and Q of Escherichia coli phage λ and the gp39 protein of Thermus thermophilus phage P23-45. These factors suppress RNAP pausing, thus inhibiting the first step of the termination pathway (4-6). The antitermination activity of N and Q, but not gp39, is enhanced by cell-encoded proteins, including transcription elongation factor NusA, which by itself stimulates termination but reverses its activity to additionally stabilize the TEC in cooperation with N and Q (5, 6). Curiously, the N, Q, gp39, and NusA proteins all target the flexible β flap domain of RNAP (2, 4...