CBR antimicrobials alter coupling between the bridge helix and the β subunit in RNA polymerase

Abstract

Bacterial RNA polymerase (RNAP) is a validated target for antibacterial drugs. CBR703 series antimicrobials allosterically inhibit transcription by binding to a conserved α helix (β′ bridge helix, BH) that interconnects the two largest RNAP subunits. Here we show that disruption of the BH-β subunit contacts by amino-acid substitutions invariably results in accelerated catalysis, slowed-down forward translocation and insensitivity to regulatory pauses. CBR703 partially reverses these effects in CBR-resistant RNAPs while inhibiting catalysis and promoting pausing in CBR-sensitive RNAPs. The differential response of variant RNAPs to CBR703 suggests that the inhibitor binds in a cavity walled by the BH, the β′ F-loop and the β fork loop. Collectively, our data are consistent with a model in which the β subunit fine tunes RNAP elongation activities by altering the BH conformation, whereas CBRs deregulate transcription by increasing coupling between the BH and the β subunit.

CBR antimicrobials inhibit RNA polymerase via at least two bridge-helix cap-mediated effects on nucleotide addition

“…Such an effect of the BH could stabilize the transition state additively with nucleotide positioning by TL folding and thus could contribute to catalysis in WT-RNAP, although we cannot currently rule out the possibility that a slow BH-dependent effect on catalysis is unmasked in ΔTL-RNAP. It also is possible that a TL-or BH-dependent effect contributes on the relevant time scale at some template positions more than others, which could explain why CBR compounds affect WT-RNAP at some template positions like pause sites more than others (6,21). Indeed, we observed more uniform effects of CBR compounds in ΔTL-RNAP than WT-RNAP (e.g., SI Appendix, Fig.…”

“…5). The CBR compounds do not compete with substrate binding and also do not principally affect translocation (6,21), reflecting an allosteric inhibitory mechanism.…”

“…As noted by Malinen et al (21), in all of the available E. coli RNAP structures [which, to date, all come from the same crystal form (24)(25)(26)], the side chain of the BH residue β′H777 points toward the C terminus of the BH and stacks on β′P750 at the tip of the F loop (Figs. 2 and 3C).…”

“…The genetically defined CBR site is ∼31 Å from the RNAP active site Mg 2+ ion and is also distinct from the binding sites of other well-characterized bacterial RNAP inhibitors (SI Appendix, Table S1). Inhibition of RNAP by the CBR compounds appears to be mechanistically distinct as well; the CBR compounds allosterically inhibit known catalytic activities of the RNAP active site preferentially at pause sites but have lesser to no effects on translocation (6,21).…”

Bacterial Transcription as a Target for Antibacterial Drug Development

“…2) consists of small molecules comprising two linked aromatic rings, isolated by screening a chemical compound library to inhibit E. coli transcription (106). CBR703, together with CBR9379 and CBR9393, demonstrated the ability to inhibit transcription elongation by stabilizing elongation complex isomerization and slowing translocation (107). The inhibitory activity of these compounds is proposed to be due to an allosteric effect that prevents TL folding, mediated via the F-loop, and the inhibition of BH movement at its N-terminal hinge (108).…”