The RNA polymerase clamp interconverts dynamically among three states and is stabilized in a partly closed state by ppGpp

Abstract

RNA polymerase (RNAP) contains a mobile structural module, the ‘clamp,’ that forms one wall of the RNAP active-center cleft and that has been linked to crucial aspects of the transcription cycle, including promoter melting, transcription elongation complex stability, transcription pausing, and transcription termination. Using single-molecule FRET on surface-immobilized RNAP molecules, we show that the clamp in RNAP holoenzyme populates three distinct conformational states and interconvert between these states on the 0.1–1 s time-scale. Similar studies confirm that the RNAP clamp is closed in open complex (RPO) and in initial transcribing complexes (RPITC), including paused initial transcribing complexes, and show that, in these complexes, the clamp does not exhibit dynamic behaviour. We also show that, the stringent-response alarmone ppGpp, which reprograms transcription during amino acid starvation stress, selectively stabilizes the partly-closed-clamp state and prevents clamp opening; these results raise the possibility that ppGpp controls promoter opening by modulating clamp dynamics.

TFS and Spt4/5 accelerate transcription through archaeal histone‐based chromatin

“…Spt4–Spt5 binds directly to RNAP and stabilizes a closed‐clamp configuration that facilitates elongation and TEC stability (Wada et al ., ; Grohmann et al ., ; Martinez‐Rucobo et al ., ; Guo et al ., ; Bernecky et al ., ; Ehara et al ., ; Kang et al ., ; Vos et al ., ). As pausing and subsequent backtracking can be influenced by inter‐domain movements of RNAP (Martinez‐Rucobo et al ., ; Chakraborty et al ., ; Weixlbaumer et al ., ; Hein et al ., ; Jun et al ., ; Blombach et al ., ; Feklistov et al ., ; Kang et al ., ; Duchi et al ., ), we sought to determine whether Spt4–Spt5 binding to RNAP would reduce pausing or accelerate transcription on protein‐free and histone‐bound templates. Addition of either Spt4 or Spt5 alone (Fig.…”

“…The inability of TFS DE-AA to properly donate acidic residues to the active site of RNAP abrogates its function as a cleavage stimulatory factor. Backtracking can result from extended pausing (Nudler, 2012;Imashimizu et al, 2013;Weixlbaumer et al, 2013;Hein et al, 2014;Imashimizu et al, 2015;James et al, 2017;Lerner et al, 2016;Gabizon et al, 2018), and the configuration of mobile-domains of RNAP is known to modulate the propensity to pause and the duration of pausing Martinez-Rucobo et al, 2011;Chakraborty et al, 2012;Weixlbaumer et al, 2013;Hein et al, 2014;Jun et al, 2014;Schulz et al, 2016;Sheppard et al, 2016;Bernecky et al, 2017;Feklistov et al, 2017;Kang et al, 2017;Duchi et al, 2018). We thus examined whether addition of Spt4 and/or Spt5 would influence the efficiency of RNA cleavage, with transcript cleavage also serving as a proxy for the propensity to, and depth of backtracking.…”

“…[Colour figure can be viewed at wileyonlinelibrary.com] 2018; Vos et al, 2018). As pausing and subsequent backtracking can be influenced by inter-domain movements of RNAP (Martinez-Rucobo et al, 2011;Chakraborty et al, 2012;Weixlbaumer et al, 2013;Hein et al, 2014;Jun et al, 2014;Blombach et al, 2016;Feklistov et al, 2017;Kang et al, 2017;Duchi et al, 2018), we sought to determine whether Spt4-Spt5 binding to RNAP would reduce pausing or accelerate transcription on protein-free and histone-bound templates. Addition of either Spt4 or Spt5 alone (Fig.…”

Closing and opening of the RNA polymerase trigger loop

“…1b). We used a procedure comprising: (i) incorporation of the fluorescent probe DyLight 550, serving as donor, in the RNAP TL, by use of unnatural-amino-acid mutagenesis and Staudinger ligation (37)(38)(39)(40); (ii) incorporation of the fluorescent probe Alexa647, serving as acceptor, in the template strand of a nucleicacid scaffold comprising a template-strand DNA oligonucleotide, a non-template strand DNA oligonucleotide, and a non-extendable, 3'-deoxyribonucleotide-containing RNA oligonucleotide; (iii) assembly and analysis of a doubly labelled transcription elongation complex (TEC) from the resulting labelled RNAP and labelled nucleic-acid scaffold; (iv) immobilization of the doubly labelled TEC, through a hexahistidine tag on RNAP on a surface functionalized with anti-hexahistidine tag-antibody;…”

“…To monitor the distance between fluorescent probes in the resulting donor-acceptor labelled elongation complexes, we used total internal reflection microscopy with alternating laser excitation (TIRF-ALEX) (37,39,40,(42)(43)(44)(45) and quantified smFRET from single TECs immobilized, through a hexahistidine tag on RNAP, on anti-hexahistidine-tag-antibody-functionalized glass cover slips. TIRF-ALEX allows filtering of data to identify only single molecules that contain both a donor and an acceptor, eliminating complications due to incomplete labelling and imperfect reconstitution of TECs (37,39,40,(42)(43)(44)(45). The results provide equilibrium population distributions of apparent smFRET efficiency, E*.…”