Mycobacterial HelD is a nucleic acids-clearing factor for RNA polymerase

Abstract

SUMMARYRNA synthesis is central to life, and RNA polymerase depends on accessory factors for recovery from stalled states and adaption to environmental changes. Here we investigated the mechanism by which a helicase-like factor HelD recycles RNA polymerase. We report a cryo-EM structure of an unprecedented complex between the Mycobacterium smegmatis RNA polymerase and HelD. The crescent-shaped HelD simultaneously penetrates deep into two RNA polymerase channels that are responsible for DNA binding and substrate delivery to the active site, thereby locking RNA polymerase in an inactive state. We show that HelD prevents non-specific interactions between RNA polymerase and DNA and dissociates transcription elongation complexes, but does not inhibit RNA polymerase binding to the initiation σ factor. The liberated RNA polymerase can either stay dormant, sequestered by HelD, or upon HelD release, restart transcription. Our results provide insights into the architecture and regulation of the highly medically-relevant mycobacterial transcription machinery and define HelD as a clearing factor that removes undesirable nucleic acids from RNA polymerase.

HelR is a helicase-like protein that protects RNA polymerase from rifamycin antibiotics

“…These proteins use the energy released from ATP hydrolysis to generate mechanical force to promote translocation along ssDNA and strand displacement. In contrast to true helicases, HelD-like enzymes lack DNA binding domains and instead use this mechanical force to remodel themselves and/or their interaction partner RNAP (Kouba et al, 2020;Newing et al, 2020;Pei et al, 2020). We demonstrated that ATP hydrolysis is essential for the resistance activity of HelRSv but that HelRSv does not inactivate the antibiotic.…”

“…Experiments with this probe revealed that HelRSv could indeed displace rifamycins from RNAP, confirming our hypothesis that HelRSv functions as a protection protein. Structural data from HelRMs implies that HelR and DNA cannot co-exist within RNAP, suggesting that HelR can only remove rifamycins from free-RNAP (Kouba et al, 2020;Newing et al, 2020;Pei et al, 2020). Incubation of HelRSv with 1mM ATP significantly reduced labeling by RPP compared to an incubation lacking ATP, suggesting that ATP hydrolysis may drive the displacement of rifamycins.…”

“…While writing this manuscript, we became aware of three co-structures of HelD in complex with RNAP published simultaneously; two describing HelD from Bacillus subtilis (HelDBs) and the third from Mycobacterium smegmatis, which has 35% amino acid identity with HelRSv (Kouba et al, 2020;Newing et al, 2020;Pei et al, 2020). The M. smegmatis protein is referred to as HelDMs and is purported to be functionally equivalent to HelDBs.…”

“…They both "wedge" open the β' Clamp weakening the interaction between RNAP and DNA and project appendages deep into RNAP. Additionally, both proteins possess a secondary channel arm (SCA) which are structurally similar to transcription factors such as GreA/B (Kouba et al, 2020;Pei et al, 2020). The SCA in HelDBs extends into and occupies the active site.…”

“…Somewhat paradoxically, HelDBs can stimulate transcription in multiple round assays by removing stalled transcription complexes from template DNA. Interestingly HelRMs has been shown to possess the ability to remove stalled elongating complexes, but there is no reported evidence that this protein can stimulate transcription in vitro (Kouba et al, 2020). We first added HelRSv in increasing concentrations to determine if this protein had any general effects on in vitro transcription (Figure 3E).…”