Advances in bacterial promoter recognition and its control by factors that do not bind DNA

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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%

Section: Discussionmentioning

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 DNA sites for CAP and ␣CTD were replaced by consensus DNA sites for CAP and ␣CTD (AAATGTGATCTA-GATCACATTT and AAAAAA), as in (8). The Ϫ10 element was replaced by a consensus Ϫ10 element (TATAAT), and the discriminator element was replaced by a consensus-type discriminator element (CGC) (19). Positions Ϫ11 to ϩ2 were made noncomplementary to create an artificial transcription bubble (Fig.…”

Section: Design Assembly and Imaging Of A Class I Cap-rnap-promotermentioning

confidence: 99%

Three-dimensional EM structure of an intact activator-dependent transcription initiation complex

Hudson

Quispe

Lara-González³

et al. 2009

We present the experimentally determined 3D structure of an intact activator-dependent transcription initiation complex comprising the Escherichia coli catabolite activator protein (CAP), RNA polymerase holoenzyme (RNAP), and a DNA fragment containing positions ؊78 to ؉20 of a Class I CAP-dependent promoter with a CAP site at position ؊61.5 and a premelted transcription bubble. A 20-Å electron microscopy reconstruction was obtained by iterative projection-based matching of single particles visualized in carbonsandwich negative stain and was fitted using atomic coordinate sets for CAP, RNAP, and DNA. The structure defines the organization of a Class I CAP-RNAP-promoter complex and supports previously proposed interactions of CAP with RNAP ␣ subunit C-terminal domain (␣CTD), interactions of ␣CTD with 70 region 4, interactions of CAP and RNAP with promoter DNA, and phased-DNAbend-dependent partial wrapping of DNA around the complex. The structure also reveals the positions and shapes of species-specific domains within the RNAP ␤ , ␤, and 70 subunits.catabolite activator protein ͉ deoxyribonucleic acid ͉ RNA polymerase holoenzyme ͉ gene regulation ͉ electron microscopy

“…10 and 11, is modulated by the protein DksA, which binds in the RNAP secondary channel. Binding of DksA or (p)ppGpp to the RNAP alone or together deceases the kinetic stability (i.e., lifetime or longevity) of open complexes and causes decreased transcription from promoters that form short-lived open complexes (e.g., promoters for ribosomal RNA synthesis) and increased transcription from promoters that form long-lived open complexes but bind RNAP weakly (e.g., promoters for some amino acid biosynthetic operons) (11). The reduction in open complex lifetime caused by (p)ppGpp and DksA are thought to redistribute RNAP away from rRNA transcription units to other genes, such as those required for amino acid prototrophy.…”

mentioning

confidence: 99%

RNA polymerase mutants found through adaptive evolution reprogram Escherichia coli for optimal growth in minimal media

Conrad¹,

Frazier

Joyce

et al. 2010

227

208

Specific small deletions within the rpoC gene encoding the β′-subunit of RNA polymerase (RNAP) are found repeatedly after adaptation of Escherichia coli K-12 MG1655 to growth in minimal media. Here we present a multiscale analysis of these mutations. At the physiological level, the mutants grow 60% faster than the parent strain and convert the carbon source 15-35% more efficiently to biomass, but grow about 30% slower than the parent strain in rich medium. At the molecular level, the kinetic parameters of the mutated RNAP were found to be altered, resulting in a 4-to 30-fold decrease in open complex longevity at an rRNA promoter and a ∼10-fold decrease in transcriptional pausing, with consequent increase in transcript elongation rate. At a genome-scale, systems biology level, gene expression changes between the parent strain and adapted RNAP mutants reveal large-scale systematic transcriptional changes that influence specific cellular processes, including strong down-regulation of motility, acid resistance, fimbria, and curlin genes. RNAP genome-binding maps reveal redistribution of RNAP that may facilitate relief of a metabolic bottleneck to growth. These findings suggest that reprogramming the kinetic parameters of RNAP through specific mutations allows regulatory adaptation for optimal growth in new environments.kinetics | stringent response | transcription