TY - JOUR TI - The mechanism of variability in transcription start site selection AU - Yu, Libing AU - Winkelman, Jared T AU - Pukhrambam, Chirangini AU - Strick, Terence R AU - Nickels, Bryce E AU - Ebright, Richard H A2 - Ha, Taekjip VL - 6 PY - 2017 DA - 2017/11/23 SP - e32038 C1 - eLife 2017;6:e32038 DO - 10.7554/eLife.32038 UR - https://doi.org/10.7554/eLife.32038 AB - 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. KW - RNA polymerase KW - scrunching KW - protein-DNA photocrosslinking KW - p-benzoyl-L-phenylalanine (Bpa) KW - single-molecule biophysics KW - magnetic tweezers JF - eLife SN - 2050-084X PB - eLife Sciences Publications, Ltd ER -