Rifampicin (Rif) is one of the most potent and broad spectrum antibiotics against bacterial pathogens and is a key component of anti-tuberculosis therapy, stemming from its inhibition of the bacterial RNA polymerase (RNAP). We determined the crystal structure of Thermus aquaticus core RNAP complexed with Rif. The inhibitor binds in a pocket of the RNAP beta subunit deep within the DNA/RNA channel, but more than 12 A away from the active site. The structure, combined with biochemical results, explains the effects of Rif on RNAP function and indicates that the inhibitor acts by directly blocking the path of the elongating RNA when the transcript becomes 2 to 3 nt in length.

RNA chain elongation is a highly processive and accurate process that is finely regulated by numerous intrinsic and extrinsic signals. Here we describe a general mechanism that governs RNA polymerase (RNAP) movement and response to regulatory inputs such as pauses, terminators, and elongation factors. We show that E.coli RNAP moves by a complex Brownian ratchet mechanism, which acts prior to phosphodiester bond formation. The incoming substrate and the flexible F bridge domain of the catalytic center serve as two separate ratchet devices that function in concert to drive forward translocation. The adjacent G loop domain controls F bridge motion, thus keeping the proper balance between productive and inactive states of the elongation complex. This balance is critical for cell viability since it determines the rate, processivity, and fidelity of transcription.

An 8-9 bp RNA-DNA hybrid in the transcription elongation complex is essential for keeping the RNA 3' terminus engaged with the active site of E. coli RNA polymerase (RNAP). Destabilization of the hybrid leads to detachment of the transcript terminus, RNAP backtracking, and shifting of the hybrid upstream. Eventually, the exposed 3' segment of RNA can be removed through transcript cleavage. At certain sites, cycles of unwinding-rewinding of the hybrid are coupled to reverse-forward sliding of the transcription elongation complex. This explains apparent discontinuous elongation, which was previously interpreted as contraction and expansion of an RNAP molecule (inch-worming). Thus, the 3'-proximal RNA-DNA hybrid plays the dual role of keeping the active site in register with the template and sensing the helix-destabilizing mismatches in RNA, launching correction through backtracking and cleavage.

The quinolones trap DNA gyrase and DNA topoisomerase IV on DNA as complexes in which the DNA is broken but constrained by protein. Early studies suggested that drug binding occurs largely along helix-4 of the GyrA (gyrase) and ParC (topoisomerase IV) proteins. However, recent X-ray crystallography shows drug intercalating between the -1 and +1 nucleotides of cut DNA, with only one end of the drug extending to helix-4. These two models may reflect distinct structural steps in complex formation. A consequence of drug-enzyme-DNA complex formation is reversible inhibition of DNA replication; cell death arises from subsequent events in which bacterial chromosomes are fragmented through two poorly understood pathways. In one pathway, chromosome fragmentation stimulates excessive accumulation of highly toxic reactive oxygen species that are responsible for cell death. Quinolone resistance arises stepwise through selective amplification of mutants when drug concentrations are above the MIC and below the MPC, as observed with static agar plate assays, dynamic in vitro systems, and experimental infection of rabbits. The gap between MIC and MPC can be narrowed by compound design that should restrict the emergence of resistance. Resistance is likely to become increasingly important, since three types of plasmid-borne resistance have been reported.

The path of the nucleic acids through a transcription elongation complex was tracked by mapping cross-links between bacterial RNA polymerase (RNAP) and transcript RNA or template DNA onto the x-ray crystal structure. In the resulting model, the downstream duplex DNA is nestled in a trough formed by the beta' subunit and enclosed on top by the beta subunit. In the RNAP channel, the RNA/DNA hybrid extends from the enzyme active site, along a region of the beta subunit harboring rifampicin resistance mutations, to the beta' subunit "rudder." The single-stranded RNA is then extruded through another channel formed by the beta-subunit flap domain. The model provides insight into the functional properties of the transcription complex.

Coliphage HK022 Nun blocks superinfection by coliphage λ by stalling RNA polymerase (RNAP) translocation specifically on λ DNA. To provide a structural framework to understand how Nun blocks RNAP translocation, we determined structures of Escherichia coli RNAP ternary elongation complexes (TECs) with and without Nun by single-particle cryo-electron microscopy. Nun fits tightly into the TEC by taking advantage of gaps between the RNAP and the nucleic acids. The C-terminal segment of Nun interacts with the RNAP β and β’ subunits inside the RNAP active site cleft as well as with nearly every element of the nucleic acid scaffold, essentially crosslinking the RNAP and the nucleic acids to prevent translocation, a mechanism supported by the effects of Nun amino acid substitutions. The nature of Nun interactions inside the RNAP active site cleft suggests that RNAP clamp opening is required for Nun to establish its interactions, explaining why Nun acts on paused TECs.DOI: http://dx.doi.org/10.7554/eLife.25478.001

In DNA-dependent RNA polymerases, reactions of RNA synthesis and degradation are performed by the same active center (in contrast to DNA polymerases in which they are separate). We propose a uni®ed catalytic mechanism for multisubunit RNA polymerases based on the analysis of its 3¢±5¢ exonuclease reaction in the context of crystal structure. The active center involves a symmetrical pair of Mg 2+ ions that switch roles in synthesis and degradation. One ion is retained permanently and the other is recruited ad hoc for each act of catalysis. The weakly bound Mg 2+ is stabilized in the active center in different modes depending on the type of reaction: during synthesis by the b,g-phosphates of the incoming substrate; and during hydrolysis by the phosphates of a non-base-paired nucleoside triphosphate. The latter mode de®nes a transient, non-speci®c nucleoside triphosphate-binding site adjacent to the active center, which may serve as a gateway for polymerization of substrates.

Summary Transcription in human mitochondria is carried out by a single-subunit, T7-like RNA polymerase assisted by several auxiliary factors. We demonstrate that an essential initiation factor, TFB2, forms a network of interactions with DNA near the transcription start site and facilitates promoter melting but may not be essential for promoter recognition. Unexpectedly, catalytic autolabeling reveals that TFB2 interacts with the priming substrate, suggesting that TFB2 acts as a transient component of the catalytic site of the initiation complex. Mapping of TFB2 identifies a region of its N-terminal domain that is involved in simultaneous interactions with the priming substrate and the templating (+1) DNA base. Our data indicate that the transcriptional machinery in human mitochondria has evolved into a system that combines features inherited from self-sufficient, T7-like RNA polymerase and those typically found in systems comprising cellular multi-subunit polymerases, and provide insights into the molecular mechanisms of transcription regulation in mitochondria.