Massively Systematic Transcript End Readout, “MASTER”: Transcription Start Site Selection, Transcriptional Slippage, and Transcript Yields

Vvedenskaya, Irina O.; Zhang, Yuanchao; Goldman, Seth R.; Valenti, Anna; Visone, Valeria; Taylor, Deanne; Ebright, Richard H.; Nickels, Bryce E.

doi:10.1016/j.molcel.2015.10.029

Cited by 73 publications

(138 citation statements)

References 26 publications

(34 reference statements)

Supporting

Mentioning

129

Contrasting

Order By: Relevance

“…In separate studies in which all possible DNA sequences between the -10 hexamer and the TSS were examined in vitro, as well as the distance between the leading edge and trailing edge of the promoter complex, it was concluded that TSS position correlates with scrunching state and is dependent on the absence of a σ 1.2 interaction with the discriminator (26,27). These data are consistent with the results reported here.…”

Section: Discussionsupporting

confidence: 87%

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

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard 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...

show abstract

Section: Discussionsupporting

confidence: 87%

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

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…To define the contribution of sequence-specific RNAP-G CRE interactions to TSS selection, we used a high-throughput sequencing-based methodology termed massively systematic transcript end readout (MASTER) (11). MASTER entails the construction of a template library that contains up to 4 10 (∼1,000,000) bar-coded sequences, production of RNA transcripts from the template library in vitro or in vivo, and analysis of transcript ends using highthroughput sequencing (11,13).…”

Section: Resultsmentioning

confidence: 99%

“…MASTER entails the construction of a template library that contains up to 4 10 (∼1,000,000) bar-coded sequences, production of RNA transcripts from the template library in vitro or in vivo, and analysis of transcript ends using highthroughput sequencing (11,13).…”

Section: Resultsmentioning

confidence: 99%

“…We previously have proposed that variability in TSS selection is mediated by variability in the size of the unwound transcription bubble (Fig. S1A) (11)(12)(13). According to this model, RPo generally contains a 13-bp unwound transcription bubble that places the template-strand nucleotide 7-bp downstream of the −10 element in the i site and places the templatestrand nucleotide 8-bp downstream of the −10 element in the i+1 site ( Fig.…”

mentioning

confidence: 99%

“…The position of the TSS relative to the position of the promoter −10 element is variable (5)(6)(7)(8)(9)(10)(11). TSS selection preferentially occurs at the position 7-bp downstream of the promoter −10 element, but can occur over a range of at least five positions, encompassing the positions 6-, 7-, 8-, 9-, or 10-bp downstream of the promoter −10 element.…”

mentioning

confidence: 99%

See 2 more Smart Citations

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

Vvedenskaya

Vahedian-Movahed

Zhang

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

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...

show abstract

Open complex DNA scrunching: A key to transcription start site selection and promoter escape

Winkelman

Gourse

2017

BioEssays

View full text Add to dashboard Cite

Summary Bacterial RNA polymerase-promoter open complexes can exist in a range of states in which the leading edge of the enzyme moves but the trailing edge does not, a phenomenon we refer to as “open complex scrunching”. Here we describe how open complex scrunching can determine the position of the transcription start site for some promoters, modulate the level of expression, and potentially could be targeted by factors to regulate transcription. We suggest that open complex scrunching at the extraordinarily active ribosomal RNA promoters might have evolved to initiate transcription at an unusual position relative to the core promoter elements in order to maximize the rate of promoter escape.

show abstract

Massively Systematic Transcript End Readout, “MASTER”: Transcription Start Site Selection, Transcriptional Slippage, and Transcript Yields

Cited by 73 publications

References 26 publications

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

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

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

Open complex DNA scrunching: A key to transcription start site selection and promoter escape

Contact Info

Product

Resources

About