Fine structure of the promoter–σ region 1.2 interaction

Self Cite

Most Escherichia coli promoters initiate transcription with a purine 7 or 8 nt downstream from the -10 hexamer, but some promoters, including the ribosomal RNA promoter rrnB P1, start 9 nt from the -10 element. We identified promoter and RNA polymerase determinants of this noncanonical rrnB P1 start site using biochemical and genetic approaches including mutational analysis of the promoter, Fe 2+ cleavage assays to monitor template strand positions near the active-site, and Bpa cross-linking to map the path of open complex DNA at amino acid and nucleotide resolution. We find that mutations in several promoter regions affect transcription start site (TSS) selection. In particular, we show that the absence of strong interactions between the discriminator region and σ region 1.2 and between the extended -10 element and σ region 3.0, identified previously as a determinant of proper regulation of rRNA promoters, is also required for the unusual TSS. We find that the DNA in the single-stranded transcription bubble of the rrnB P1 promoter complex expands and is "scrunched" into the active site channel of RNA polymerase, similar to the situation in initial transcribing complexes. However, in the rrnB P1 open complex, scrunching occurs before RNA synthesis begins. We find that the scrunched open complex exhibits reduced abortive product synthesis, suggesting that scrunching and unusual TSS selection contribute to the extraordinary transcriptional activity of rRNA promoters by increasing promoter escape, helping to offset the reduction in promoter activity that would result from the weak interactions with σ. T o initiate transcription, RNA polymerase (RNAP) and promoter DNA participate in a multistep binding reaction that results in unwinding of at least one turn of DNA and placement of the template strand into the active site. Once positioned, the initiating nucleoside triphosphate and the second NTP pair with the template, and the first phosphodiester bond is catalyzed.There is considerable information about the structure of open complexes and the position of the transcription start site (TSS) for consensus promoters and engineered scaffolds. However, perfect consensus promoters are not actually found in wild-type Escherichia coli cells, and little is known about TSS selection on native promoters and about how variation in promoter sequence affects TSS position. Comparison of the TSS for natural and synthetic Eσ 70 -dependent promoters indicates that a purine 7-8 bp downstream from the last base in the -10 element is typically used for initiation, and in some cases, two or more adjacent positions in an individual promoter are used (1-3). A pyrimidine at nontemplate strand position −1 is preferred (Fig. 1A), because the corresponding purine on the template strand makes favorable stacking interactions with the initiating NTP (4). Transcripts initiating as far as 12 bp downstream from the -10 hexamer have been reported but are very uncommon (5, 6). Changes in nucleotide concentrations alter the TSS at some promoters, and t...

Section: Resultsmentioning

confidence: 99%

Open complex scrunching before nucleotide addition accounts for the unusual transcription start site of E. coli ribosomal RNA promoters

Winkelman

Chandrangsu

Ross

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%

“…Substitutions of the discriminator bases and the σ and β residues that interact with the NT strand lead to decreases in open complex stability (11,14,16). To test whether the loss of the GL-NT DISC contacts observed in the RPo structure (Fig.…”

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...

“…1B). A comparable mutation in the ribosomal promoter rrnB P1 has been shown to increase RPo half-life via interaction with 70 region 1.2 residue M102 and, in addition, the absence of region 1.1 increases the detection of this contact (22,33). P min11 is also inhibited by region 1.1 (Fig.…”

Section: Effectmentioning

confidence: 94%

“…Finally, the DNA around the ϩ1 site begins to melt, and 70 region 2.3 contacts single-stranded (ss)DNA bases at positions Ϫ10 through Ϫ7 on the nontemplate strand. For some promoters, contract(s) between residues in 70 region 1.2 and ss bases at Ϫ5 and Ϫ6 occurs also (22,33). The protein/ssDNA interactions stabilize the polymerase/promoter complex, allowing the template strand to descend into the active site of core and the dsDNA downstream to fully enter the downstream channel.…”

mentioning

confidence: 99%

The promoter spacer influences transcription initiation via σ ⁷⁰ region 1.1 of Escherichia coli RNA polymerase

Hook-Barnard

Hinton

2009

Transcription initiation is a dynamic process in which RNA polymerase (RNAP) and promoter DNA act as partners, changing in response to one another, to produce a polymerase/promoter open complex (RPo) competent for transcription. In Escherichia coli RNAP, region 1.1, the N-terminal 100 residues of 70 , is thought to occupy the channel that will hold the DNA downstream of the transcription start site; thus, region 1.1 must move from this channel as RPo is formed. Previous work has also shown that region 1.1 can modulate RPo formation depending on the promoter. For some promoters region 1.1 stimulates the formation of open complexes; at the P minor promoter, region 1.1 inhibits this formation. We demonstrate here that the AT-rich P minor spacer sequence, rather than promoter recognition elements or downstream DNA, determines the effect of region 1.1 on promoter activity. Using a P minor derivative that contains good 70 -dependent DNA elements, we find that the presence of a more GC-rich spacer or a spacer with the complement of the P minor sequence results in a promoter that is no longer inhibited by region 1.1. Furthermore, the presence of the Pminor spacer, the GC-rich spacer, or the complement spacer results in different mobilities of promoter DNA during gel electrophoresis, suggesting that the spacer regions impart differing conformations or curvatures to the DNA. We speculate that the spacer can influence the trajectory or flexibility of DNA as it enters the RNAP channel and that region 1.1 acts as a ''gatekeeper'' to monitor channel entry.