Transcription inactivation through local refolding of the RNA polymerase structure

Abstract

Structural studies of antibiotics not only provide a shortcut to medicine allowing for rational structure-based drug design, but may also capture snapshots of dynamic intermediates that become 'frozen' after inhibitor binding. Myxopyronin inhibits bacterial RNA polymerase (RNAP) by an unknown mechanism. Here we report the structure of dMyx--a desmethyl derivative of myxopyronin B--complexed with a Thermus thermophilus RNAP holoenzyme. The antibiotic binds to a pocket deep inside the RNAP clamp head domain, which interacts with the DNA template in the transcription bubble. Notably, binding of dMyx stabilizes refolding of the beta'-subunit switch-2 segment, resulting in a configuration that might indirectly compromise binding to, or directly clash with, the melted template DNA strand. Consistently, footprinting data show that the antibiotic binding does not prevent nucleation of the promoter DNA melting but instead blocks its propagation towards the active site. Myxopyronins are thus, to our knowledge, a first structurally characterized class of antibiotics that target formation of the pre-catalytic transcription initiation complex-the decisive step in gene expression control. Notably, mutations designed in switch-2 mimic the dMyx effects on promoter complexes in the absence of antibiotic. Overall, our results indicate a plausible mechanism of the dMyx action and a stepwise pathway of open complex formation in which core enzyme mediates the final stage of DNA melting near the transcription start site, and that switch-2 might act as a molecular checkpoint for DNA loading in response to regulatory signals or antibiotics. The universally conserved switch-2 may have the same role in all multisubunit RNAPs.

Figure 1. Structure of the RNAP–Myx complex

The same colour scheme is used in all figures throughout this manuscript. The σ-subunit, bridge helix, trigger loop and the remainder of the RNAP molecule are in blue, magenta, cyan and grey, respectively. The switch-2 segments in the Myx-free and Myx-bound structures are in orange and green, respectively. dMyx is in black. The Mg²⁺ ion is shown as magenta sphere. a, The overall view of the complex is shown. CC, coiled–coil; CH, clamp helices. b, Close-up view of the dMyx binding site. c, Sequence alignment of the switch-2 segment from bacterial (bsu, Bacillus subtilis; eco, E. coli; mtu, Mycobacterium tuberculosis; tt, T. thermophilus), archaeal (pfu, Pyrococcus furiosis) and yeast Saccharomyces cerevisiae pol II (scII) enzymes. Substitutions constructed in this work are shown above the sequence in green. d, Schematic drawing of the protein–dMyx interactions. The polar and van der Waals interactions are shown as solid arrows and dashed lines, respectively. The mutated residues are indicated by the red boxes. e, f, Conformations of the switch-2 segment in the Myx-free (e) and Myx-bound (f) holo-RNAP structures. In the panels d and e the E. coli residue numbers are shown in blue.

Figure 2. Effect of RNAP mutations on dMyx activity

The half-maximal inhibitory concentration (IC₅₀) values were measured in vitro with purified RNAP variants (see Methods). The data is for all variants tested in this study. The IC₅₀ could not be determined for the highly resistant β’ K345A variant. The T. thermophilus residue numbers are shown in brackets. Errors are standard deviations of the best fit estimates for IC₅₀ and were calculated by nonlinear regression of the RNA synthesis measurements versus dMyx concentration assuming a hyperbolic dependence.

Figure 3. A mechanism of the dMyx action

a, Myx alters the contacts between RNAP and λP_R promoter DNA. a, A linear DNA fragment encompassing positions −81 to +70 of the λP_R promoter was generated by polymerase chain reaction (PCR); the non-template DNA strand was end-labelled with [³²P]-γATP (see Methods). The sequence from −44 to +23 is shown. The −35 and −10 hexamers are indicated by black boxes, the start site (+1) is shown by a black dot. The top panel shows probing of the non-template strand by piperidine-induced cleavage of the permanganate-modified T residues. Reactivities of −10, −4, −3 and +3 residues (quantification described in Methods) are shown to the left of the gel and summarized above the promoter sequence where black and white arrows indicate high and low reactivity, respectively. The bottom panel shows protection of the non-template DNA strand from DNaseI digestion. The footprint boundaries in the promoter region shown are indicated on the gel and by black (RNAP alone) and white (RNAP with the inhibitor) bars below the promoter sequence; the dideoxy-sequencing ladder is shown for reference. In the gels shown, independent reaction repeats were analysed for consistency. b, Schematic drawing of the putative mechanism of the dMyx action. dwDNA, downstream DNA.

Figure 4. Mutations in switch-2 affect the open complex formation

Accessibility of the non-template DNA (blue) strand residues to permanganate modification probed as in Fig. 3a. Wild-type and mutant RNAPs differ in their patterns of reactivity in the absence of dMyx (top traces) but are nearly identical in the presence of 10 μM dMyx (bottom traces). Notably, β’Δ309–325 that removes the entire rudder loop (which is inserted in the same helix as switch-2, but is unlikely to interfere with the nucleic acids) has no effect on DNA melting, suggesting that a melting defect of a different rudder deletion might be due to changes in the adjacent switch-2 instead.