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of the RNAP active-center cleft and that has been linked to crucial aspects of the transcription cycle, including was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/278838 doi: bioRxiv preprint first posted online Mar. 8, 2018; 2 SIGNIFICANCE STATEMENTThe clamp forms a pincer of the RNA polymerase "crab-claw" structure, and adopts many conformations with poorly understood function and dynamics. By measuring distances within single surface-attached molecules, we observe directly the motions of the clamp and show that it adopts an open, a closed, and a partly closed state; the last state is stabilized by a sensor of bacterial starvation, linking the clamp conformation to the mechanisms used by bacteria to counteract stress. We also show that the clamp remains closed in many transcription steps, as well as in the presence of a specific antibiotic. Our approach can monitor clamp motions throughout transcription and offers insight on how antibiotics can stop pathogens by blocking their RNA polymerase movements. INTRODUCTIONRNA polymerase (RNAP) is the main molecular machine responsible for transcription. In bacteria, RNAP is a multi-subunit protein with an overall shape that resembles a crab claw. Comparison of high-resolution structures of RNAP in different crystal lattices (1-5) reveals that one of the two "pincers" of the RNAP crab claw, the "clamp", can adopt different orientations relative to the rest of RNAP, due to up to ~20 o swinging movements of the clamp about a molecular hinge at its base, the RNAP "switch region" (5-9).The observation of multiple RNAP clamp conformations suggested that the RNAP clamp is a mobile structural module and raised the possibility that RNAP clamp movements may be functionally important for transcription (5-9). In particular, it has been proposed that the RNAP clamp opening may be important for loading DNA into the RNAP active-center cleft and that RNAP clamp closing may be important for retaining DNA inside the RNAP active-center cleft and providing high transcription-complex stability in late stages of transcription initiation and in transcription elongation (5-12).Single-molecule FRET (smFRET) studies assessing RNAP clamp conformation in solution in freely diffusing single molecules of Escherichia coli RNAP confirmed that the RNAP clamp adopts different conformational states in solution; defined equilibrium population distributions of RNAP clamp states in RNAP core enzyme, RNAP holoenzyme, transcription initiation complexes, and transcription elongation complexes; and demonstrated effects of RNAP inhibitors that interact with the RNAP switch region on RNAP clamp conformation (9). However, because those smFRET studies analyzed freely diffusing single molecules--for which it is difficult to monitor individual single molecules over timescale...

RNA polymerase clamp conformational dynamics: long-lived states and modulation by crowding, cations, and nonspecific DNA binding

“…Oligonucleotides were purchased from Sigma Aldrich and annealed in hybridization buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 1 mM EDTA). The sequence for the non-specific DNA was generated from the lacCONS promoter sequence (15) by full substitution of the -35 and -10 elements, as follows:…”

“…RNAP complex with non-specific DNA were prepared by mixing 20 nM of biotinylated-non-specific dsDNA with 50 nM labelled RNAP for 5 min at 37ºC in KG7 buffer (40 mM HEPES-NaOH, pH 7.0, 100 mM potassium glutamate, 10 mM MgCl2, 1 mM dithiothreitol and 5% glycerol). Figure 3B, a biotin-PEG-passivated glass surface was prepared, functionalized with neutravidin and treated with biotinylated anti-hexahistidine monoclonal antibody (Qiagen) as described (15). RNAP immobilisation was performed by adding 100 pM solution of labelled RNAP holoenzyme to the PEGylated surfaces with biotinylated anti-hexahistidine monoclonal antibody (15) for 5 min at 22C in KG7 buffer (40 mM HEPES-NaOH, pH 7.0, 100 mM potassium glutamate, 10 mM MgCl2, 1 mM dithiothreitol, 5% glycerol).…”

“…We observed that, in RNAP holoenzyme, the clamp populates three distinct conformations: an open clamp, a partly closed clamp, and a closed clamp conformation (13,15,16). We also showed that the clamp switches among these three short-lived conformational states on the millisecond time scale and can be trapped in any one of these three conformations by the RNAP inhibitors myxopyronin, corallopyronin, ripostatin, fidaxomicin (lipiarmycin), and ppGppp (13,15,16). Additionally, we observed that the clamp adopts exclusively or nearly exclusively, a closed conformation in the RNAP-promoter open complex (RPo), RNAP-promoter initial transcribing complexes (RPitc), and RNAP-DNA elongation complexes (RDe) (13,15).…”

“…We first used the resulting doubly labelled RNAP derivatives to monitor clamp conformations in freely diffusing RNAP molecules in solution through smFRET with confocal alternating laser excitation microscopy (confocal ALEX; 13). We next used the doubly labelled RNAP derivatives to monitor clamp conformational dynamics in surface-immobilized RNAP molecules in solution, in real time, through smFRET with total internal reflection fluorescence ALEX microscopy (TIRF-ALEX; [15][16]. We observed that, in RNAP holoenzyme, the clamp populates three distinct conformations: an open clamp, a partly closed clamp, and a closed clamp conformation (13,15,16).…”

“…We next used the doubly labelled RNAP derivatives to monitor clamp conformational dynamics in surface-immobilized RNAP molecules in solution, in real time, through smFRET with total internal reflection fluorescence ALEX microscopy (TIRF-ALEX; [15][16]. We observed that, in RNAP holoenzyme, the clamp populates three distinct conformations: an open clamp, a partly closed clamp, and a closed clamp conformation (13,15,16). We also showed that the clamp switches among these three short-lived conformational states on the millisecond time scale and can be trapped in any one of these three conformations by the RNAP inhibitors myxopyronin, corallopyronin, ripostatin, fidaxomicin (lipiarmycin), and ppGppp (13,15,16).…”