SummaryIn bacteria, RNA polymerase (RNAP) initiates transcription by synthesizing short transcripts that are either released or extended to allow RNAP to escape from the promoter. The mechanism of initial transcription is unclear due to the presence of transient intermediates and molecular heterogeneity. Here, we studied initial transcription on a lac promoter using single-molecule fluorescence observations of DNA scrunching on immobilized transcription complexes. Our work revealed a long pause (“initiation pause,” ∼20 s) after synthesis of a 6-mer RNA; such pauses can serve as regulatory checkpoints. Region sigma 3.2, which contains a loop blocking the RNA exit channel, was a major pausing determinant. We also obtained evidence for RNA backtracking during abortive initial transcription and for additional pausing prior to escape. We summarized our work in a model for initial transcription, in which pausing is controlled by a complex set of determinants that modulate the transition from a 6- to a 7-nt RNA.

Fidaxomicin is an antibacterial drug in clinical use for treatment of Clostridium difficile diarrhea. The active ingredient of fidaxomicin, lipiarmycin A3 (Lpm), functions by inhibiting bacterial RNA polymerase (RNAP). Here we report a cryo-EM structure of Mycobacterium tuberculosis RNAP holoenzyme in complex with Lpm at 3.5-Å resolution. The structure shows that Lpm binds at the base of the RNAP "clamp." The structure exhibits an open conformation of the RNAP clamp, suggesting that Lpm traps an open-clamp state. Single-molecule fluorescence resonance energy transfer experiments confirm that Lpm traps an open-clamp state and define effects of Lpm on clamp dynamics. We suggest that Lpm inhibits transcription by trapping an open-clamp state, preventing simultaneous interaction with promoter -10 and -35 elements. The results account for the absence of cross-resistance between Lpm and other RNAP inhibitors, account for structure-activity relationships of Lpm derivatives, and enable structure-based design of improved Lpm derivatives.

Bacterial transcription is initiated after RNA polymerase (RNAP) binds to promoter DNA, melts ~14 bp around the transcription start site and forms a single-stranded “transcription bubble” within a catalytically active RNAP–DNA open complex (RPo). There is significant flexibility in the transcription start site, which causes variable spacing between the promoter elements and the start site; this in turn causes differences in the length and sequence at the 5′ end of RNA transcripts and can be important for gene regulation. The start-site variability also implies the presence of some flexibility in the positioning of the DNA relative to the RNAP active site in RPo. The flexibility may occur in the positioning of the transcription bubble prior to RNA synthesis and may reflect bubble expansion (“scrunching”) or bubble contraction (“unscrunching”). Here, we assess the presence of dynamic flexibility in RPo with single-molecule FRET (Förster resonance energy transfer). We obtain experimental evidence for dynamic flexibility in RPo using different FRET rulers and labeling positions. An analysis of FRET distributions of RPo using burst variance analysis reveals conformational fluctuations in RPo in the millisecond timescale. Further experiments using subsets of nucleotides and DNA mutations allowed us to reprogram the transcription start sites, in a way that can be described by repositioning of the single-stranded transcription bubble relative to the RNAP active site within RPo. Our study marks the first experimental observation of conformational dynamics in the transcription bubble of RPo and indicates that DNA dynamics within the bubble affect the search for transcription start sites.

Transcription initiation is a major step in gene regulation for all organisms. In bacteria, the promoter DNA is first recognized by RNA polymerase (RNAP) to yield an initial closed complex. This complex subsequently undergoes conformational changes resulting in DNA strand separation to form a transcription bubble and an RNAP-promoter open complex; however, the series and sequence of conformational changes, and the factors that influence them are unclear. To address the conformational landscape and transitions in transcription initiation, we applied single-molecule Förster resonance energy transfer (smFRET) on immobilized Escherichia coli transcription open complexes. Our results revealed the existence of two stable states within RNAP–DNA complexes in which the promoter DNA appears to adopt closed and partially open conformations, and we observed large-scale transitions in which the transcription bubble fluctuated between open and closed states; these transitions, which occur roughly on the 0.1 s timescale, are distinct from the millisecond-timescale dynamics previously observed within diffusing open complexes. Mutational studies indicated that the σ70 region 3.2 of the RNAP significantly affected the bubble dynamics. Our results have implications for many steps of transcription initiation, and support a bend-load-open model for the sequence of transitions leading to bubble opening during open complex formation.

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.

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...

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Fidaxomicin is an antibacterial drug in clinical use in treatment ofWe determined a structure of Mycobacterium tuberculosis (Mtb) RNAP σ A holoenzyme in complex with Lpm by use of cryo-EM with single particle reconstruction ( Fig. 1; Extended Data Figs.1-3). Density maps showed unambiguous density for RNAP, including the taxon-specific, 2 peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/237123 doi: bioRxiv preprint first posted online Dec. 20, 2017; Mycobacterium-specific sequence insertion 15 (β′MtbSI or "gate"), for σ conserved regions 2, 3, and 4 (σR2, σR3, and σR4), and for Lpm. The mean resolution of the structure is 3.5 Å (Extended Data Fig. 1e).Local resolution ranges from ~2.5-3.8 Å in central parts of the structure, including RNAP residues close to Lpm, to ~5-6.5 Å in peripheral parts of the structure, including β′MtbSI and σR4 (Fig. 1a; Extended Data Figs. 1f, 2a). Local B-factors range from ~0-100 Å 2 in central regions to ~300-400 Å 2 in peripheral regions (Extended Data Fig. 2b). Density maps show clear density for backbone and sidechain atoms of RNAP and individual functional groups of Lpm (Extended Data Fig. 2c-d).The cryo-EM structure shows that the Lpm binding site is located at the base of the RNAP (D248), and β′R412 (R337) (residues numbered as in Mtb RNAP and, in parentheses, as in Escherichia coli RNAP). Alanine substitutions of these RNAP residues results in Lpm-resistance, confirming the interactions are functionally relevant (Extended Data Fig. 3d).Sequence alignments show that residues of RNAP that contact Lpm are conserved inGram-positive and Gram-negative bacterial RNAP, but are not conserved in human RNAP I, II, and III (Extended Data Fig. 4a-b), accounting for the ability of Lpm to inhibit both Gram-positive and Gramnegative bacterial RNAP but not human RNAP I, II, and III (Extended Data Fig. 4c).The cryo-EM structure accounts for structure-activity relationships of Lpm analogs produced by metabolism 18 , precursor feeding 19 , mutasynthesis [20][21] , and semi-synthesis ( Fig. 1d- peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/237123 doi: bioRxiv preprint first posted online Dec. 20, 2017; Lpm), and homodichloro-orsellinyl moiety and C8" methyl, C3" chlorine, and C5" chlorine therein (d1 vs. Lpm; a2-a4 vs. Lpm; a8 vs. a6). The observation that the Lpm C4" hydroxyl is exposed to solvent at the exterior of RNAP (Extended Data Fig. 3b-c) predicts that substituents--including large substituents--may be introduced at the C4" hydroxyl oxygen without eliminating ability to inhibit RNAP, provided the H-bond-acceptor character of the oxygen is maintained. Validating this prediction, we have prepared a novel Lpm analog having benzyl appended at the C4" hydroxyl oxygen by semi-synthesis from Lpm and have found the analog to retain hig...