The mechanism of variability in transcription start site selection.

Libing Yu,Richard H Ebright,Chirangini Pukhrambam,Bryce E Nickels,Terence R Strick,Jared T Winkelman

doi:10.7554/elife.32038

Libing Yu, Richard H Ebright + Show 4 more

Open Access

https://doi.org/10.7554/elife.32038

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Journal: eLife	Publication Date: Nov 23, 2017
Citations: 24	License type: CC BY 4.0

Affiliation: Rutgers, The State University of New Jersey

Abstract

During transcription initiation, RNA polymerase (RNAP) binds to promoter DNA, unwinds promoter DNA to form an RNAP-promoter open complex (RPo) containing a single-stranded 'transcription bubble,' and selects a transcription start site (TSS). TSS selection occurs at different positions within the promoter region, depending on promoter sequence and initiating-substrate concentration. Variability in TSS selection has been proposed to involve DNA 'scrunching' and 'anti-scrunching,' the hallmarks of which are: (i) forward and reverse movement of the RNAP leading edge, but not trailing edge, relative to DNA, and (ii) expansion and contraction of the transcription bubble. Here, using in vitro and in vivo protein-DNA photocrosslinking and single-molecule nanomanipulation, we show bacterial TSS selection exhibits both hallmarks of scrunching and anti-scrunching, and we define energetics of scrunching and anti-scrunching. The results establish the mechanism of TSS selection by bacterial RNAP and suggest a general mechanism for TSS selection by bacterial, archaeal, and eukaryotic RNAP.

Highlights

During transcription initiation, RNA polymerase (RNAP) and one or more transcription initiation factor bind to promoter DNA through sequence-specific interactions with core promoter elements, unwind a turn of promoter DNA to form an RNAP-promoter open complex (RPo) containing an unwound ‘transcription bubble,’ and select a transcription start site (TSS)
We demonstrated that bacterial TSS selection in vitro exhibits the first hallmark of scrunching by defining, simultaneously, the TSS, the RNAP leading-edge position, and RNAP trailing-edge position for transcription complexes formed on a library of 106 promoter sequences (Winkelman et al, 2016a)
The results showed that the discriminator element (Haugen et al, 2006; Feklistov et al, 2006) influences TSS selection and does so through effects on sequence-specific sDNA interaction that select between two alternative paths of the DNA nontemplate strand (Winkelman et al, 2016a)

Summary

Introduction

RNA polymerase (RNAP) and one or more transcription initiation factor bind to promoter DNA through sequence-specific interactions with core promoter elements, unwind a turn of promoter DNA to form an RNAP-promoter open complex (RPo) containing an unwound ‘transcription bubble,’ and select a transcription start site (TSS). By mapping the positions of the two edges of RNA polymerase relative to DNA, they saw that RNA polymerase pulls in extra DNA when selecting a transcription start site further from its initial attachment site. In addition to these four promoter-sequence determinants, the concentrations of initiating NTPs (Sørensen et al, 1993; Liu and Turnbough, 1994; Walker and Osuna, 2002; Vvedenskaya et al, 2015; Wilson et al, 1992; Qi and Turnbough, 1995; Tu and Turnbough, 1997; Walker et al, 2004; Turnbough, 2008; Turnbough and Switzer, 2008) and DNA topology (Vvedenskaya et al, 2015) influence TSS selection. We show that TSS selection exhibits the first hallmark of scrunching in vivo, show that TSS selection exhibits the second hallmark of scrunching and antiscrunching, and define the energetics of scrunching and anti-scrunching

Results and discussion

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Materials and methods

Funding Funder National Institutes of Health