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

Abstract

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...

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

“…Increasing the distance between the −10 region and the start site is expected to weaken these contacts, facilitating promoter escape (17). In agreement with recent reports (12, 17), we found that the discriminator, but not other promoter regions, determined the differences in abortive synthesis between the T7A1 and λP R promoters (Fig.…”

“…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).…”

“…Despite forming very unstable complexes, rrnB P1 is one of the strongest promoters, in part because it does not produce abortive RNAs. C-7G substitution restores favorable interactions with σ1.2, stabilizing the rrnB P1 promoter complex ∼40-fold (15) and impeding promoter escape (17). A very stable λP R RPo with a 6-nt discriminator makes favorable contacts with σ1.2 and produces abundant abortive products; the corresponding G-5C substitution reduces its half-life 14-fold (15).…”

“…RNAP can start transcription at a range of positions relative to the −10 hexamer (27), and σ1.2-NT DISC interactions play a key role in TSS selection (17). WT rrnB P1 complexes, in which DNA is scrunched, initiate at 9A (+1), whereas the C-7G substitution or spacer insertions that reduce scrunching shift the TSS to a "standard" 6A (17). By altering the NT DISC path through RNAP, the GL also may contribute to TSS choice.…”

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

“…S1A) (TSS = 6). According to this model, any protein-DNA or proteinprotein interaction that affects the energy landscape for transcription bubble expansion or contraction (scrunching or antiscrunching) in RPo potentially could modulate TSS selection (13,14).…”