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.

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Artificial intelligence can help tackle the covid-19 pandemic, but bias and discrimination in its design and deployment risk exacerbating existing health inequity argue David Leslie and colleagues on 21 March 2021 by guest. Protected by copyright.

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...

Summary A three-step procedure comprising (i) unnatural-amino-acid mutagenesis with 4-azido-phenylalanine, (ii) Staudinger-Bertozzi ligation with a probe-phosphine derivative, and (iii) in vitro reconstitution of RNA polymerase (RNAP) enables the efficient site-specific incorporation of a fluorescent probe, a spin label, a crosslinking agent, a cleaving agent, an affinity tag, or any other biochemical or biophysical probe, at any site of interest in RNAP. Straightforward extensions of the procedure enable the efficient site-specific incorporation of two or more different probes in two or more different subunits of RNAP. We present protocols for synthesis of probe-phosphine derivatives, preparation of RNAP subunits and the transcription initiation factor σ, unnatural amino acid mutagenesis of RNAP subunits and σ, Staudinger ligation with unnatural-amino-acid-containing RNAP subunits and σ, quantitation of labelling efficiency and labelling specificity, and reconstitution of RNAP.

a b s t r a c t k-Repressor-operator sites interaction, particularly O R 1 and O R 2, is a key component of the k-genetic switch. FRET from the dansyl bound to the C-terminal domain of the protein, to the intercalated EtBr in the operator DNA indicates that the structure of the protein is more compact in the O R 2 complex than in the O R 1 complex. Fluorescence anisotropy reveals enhanced flexibility of the C-terminal domain of the repressor at fast timescales after complex formation with O R 1. In contrast, O R 2 bound repressor shows no significant enhancement of protein dynamics at these timescales. These differences are shown to be important for correct protein-protein interactions. Altered protein dynamics upon specific DNA sequence recognition may play important roles in assembly of regulatory proteins at the correct positions.