Transcription inactivation through local refolding of the RNA polymerase structure

Abstract

Structural studies of antibiotics not only provide a short cut to medicine allowing for rational structure-based drug design, but may also capture snapshots of dynamic intermediates that become ‘frozen’ after inhibitor binding1,2. Myxopyronin inhibits bacterial RNA polymerase (RNAP) by an unknown mechanism3. Here we report the structure of dMyx—a desmethyl derivative of myxopyronin B4—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 bubble5,6. Notably, binding of dMyx stabilizes refolding of the β’-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.

Structure-Based Ligand Design of Novel Bacterial RNA Polymerase Inhibitors

“…6 New opportunities to explore bacterial RNAP as a target have arisen following the 2008 discovery of the myxopyronin B 1 (MyxB) binding site (Figure 1). 7,8 MyxB is an antibiotic produced from the myxobacterium Myxococcus fulvus Mxf50, which inhibits bacterial RNAP and the growth of Gram-positive and some Gram-negative bacteria [1: IC 50 , 0.92 μM; minimum inhibitory concentration (MIC), 1 μg/mL Staphylococcus aureus]. 9,10 MyxB binds to the switch region of RNAP, where its mode of action has been proposed to involve either inhibition of the opening and closing of the β 0 subunit 8 or stabilization of the refolding of the β 0 -subunit switch-2 region, leading to an inactive conformation that cannot bind to template DNA.…”

“…9,10 MyxB binds to the switch region of RNAP, where its mode of action has been proposed to involve either inhibition of the opening and closing of the β 0 subunit 8 or stabilization of the refolding of the β 0 -subunit switch-2 region, leading to an inactive conformation that cannot bind to template DNA. 7 As bacteria have become resistant to the rifamycins, the MyxB binding site presents itself as an excellent opportunity for the design of new RNAP inhibitors as the two antibiotic binding sites are distant from each other. 8 Inspection of the available cocrystal structures 7,8 revealed that the MyxB binding site within RNAP is U-shaped and sandwiched between two hydrophobic pockets ( Figure 1a).…”

“…7 As bacteria have become resistant to the rifamycins, the MyxB binding site presents itself as an excellent opportunity for the design of new RNAP inhibitors as the two antibiotic binding sites are distant from each other. 8 Inspection of the available cocrystal structures 7,8 revealed that the MyxB binding site within RNAP is U-shaped and sandwiched between two hydrophobic pockets ( Figure 1a). MyxB fills this cavity via the location of lipophilic side chains deep within these pockets.…”

“…Using the cocrystal structure of T. thermophilus RNAP-desmyxopyronin B complex (PDB ID: 3EQL), 7 the MyxB binding site was explored using SPROUT. Given the large volume of this cavity, we focused on the region around the enecarbamate side chain, since it was narrow and offered a bound inhibitor the potential for making hydrogen-bonding interactions with residues Trp1039 and Glu1041 (Figure 1c).…”

Antibiotics trapping transcription initiation intermediates

“…Understanding transcriptional regulation requires the characterization of the intermediates and checkpoints in the initiation pathway leading to a transcriptionally active RNAP-promoter complex, generically referred to as RP o . The antibiotics lipiarmycin (Lpm) and myxopyronin (Myx), which target two distinct steps in forming RP o , [1][2][3] open up new perspectives for fundamental studies of transcriptional regulation and for medical research to identify new drug target sites. …”