How to Shut Down Transcription in Archaea during Virus Infection

Abstract

Multisubunit RNA polymerases (RNAPs) carry out transcription in all domains of life; during virus infection, RNAPs are targeted by transcription factors encoded by either the cell or the virus, resulting in the global repression of transcription with distinct outcomes for different host-virus combinations. These repressors serve as versatile molecular probes to study RNAP mechanisms, as well as aid the exploration of druggable sites for the development of new antibiotics. Here, we review the mechanisms and structural basis of RNAP inhibition by the viral repressor RIP and the crenarchaeal negative regulator TFS4, which follow distinct strategies. RIP operates by occluding the DNA-binding channel and mimicking the initiation factor TFB/TFIIB. RIP binds tightly to the clamp and locks it into one fixed position, thereby preventing conformational oscillations that are critical for RNAP function as it progresses through the transcription cycle. TFS4 engages with RNAP in a similar manner to transcript cleavage factors such as TFS/TFIIS through the NTP-entry channel; TFS4 interferes with the trigger loop and bridge helix within the active site by occlusion and allosteric mechanisms, respectively. The conformational changes in RNAP described above are universally conserved and are also seen in inactive dimers of eukaryotic RNAPI and several inhibited RNAP complexes of both bacterial and eukaryotic RNA polymerases, including inactive states that precede transcription termination. A comparison of target sites and inhibitory mechanisms reveals that proteinaceous repressors and RNAP-specific antibiotics use surprisingly common ways to inhibit RNAP function.

Keywords: RNA polymerase; antibiotics; archaea; evolution; transcription inhibition; viruses.

Figure 1

The archaeal transcription cycle. TBP, TFB and TFE form together with RNAP, the PIC on the archaeal promoter, to initiate transcription on the transcription start site (TSS). Following promoter escape, the elongation factors Spt4/5 and Elf1 associate with the RNAP forming the TEC. When encountering roadblocks, the TEC pauses, and the RNA is backtracked through the NTP-entry pore. TFS1 promotes the cleavage of the ‘excess’ of RNA in the pore, creates a new RNA 3′-end, which reactivates the TEC. Transcription termination can occur through a factor-independent or -dependent mechanisms at the transcription termination site (TTS) and utilises aCPSF1. aCPSF1 is also recruited proximal to the promoter where it can lead to premature termination. The inhibitory regulators RIP and TFS4 interfere with RNAP as it progresses through the transcription cycle as listed in the light blue box.

Figure 2

The archaeal RNAP–RIP complex. (a) The Sulfolobus acidocaldarius RNAP is shown in ribbon style with ions as spheres, zinc in medium purple, iron in red, and sulfur in yellow. RIP is highlighted in sea green with the N- and C-termini annotated, the clamp in steel blue, and the rudder in blue (pdb 7oq4). (b) Superimposition of the RNAP–RIP complex with TFIIB of the eukaryotic PIC (pdb 6gyk [48]). The archaeal RNAP is shown as surface with RIP highlighted in sea green as ribbon style, while for the eukaryotic PIC complex only TFIIB has been shown (in yellow).

Figure 3

Molecular mechanisms of TFS4 inhibition. (a) Cryo-EM structure of the RNAP–TFS4 complex (pdb 7oqy) in ribbon style with ions as spheres, zinc in medium purple, magnesium in green, iron in red, and sulphur in yellow. TFS4 is shown in magenta with the N- and C-terminal zinc ribbon domains indicated. (b) Enlargement of the TFS4 binding site shown as surface with the lobe in dark pink, the upper jaw in olive, and the rim helices in light green, while bridge helix, BH in gold, trigger loop, TL in lime, and TFS4 are in ribbon style. Zinc ions are displayed as spheres in medium purple. (c) Superimposition of the TFS4–RNAP complex (in grey) with the apo-RNAP (in dark blue from pdb 7ok0). The red arrows indicate the direction of the downwards swinging of the jaw and clamp, as well as the stretch of the bridge helix and the extent of the DNA-binding channel opening.

Figure 4

RNAP-specific antibiotics that are reminiscent of RIP and TFS4 action. Schematic illustration of the RNAP target sites for a selection of antibiotics and inhibitors. The RNAPs are shown as grey surfaces in transparency, the antibiotics and the inhibitors TFS4 and RIP are shown as surface representation using a colour code based on closely related binding sites: medium purple for the NTP-entry channel, orange for the RNA binding site, light sky blue for the BH/TL site, and sea green for the clamp region. All bacterial structures were superimposed against subunit beta; pdb codes: 5uh6 (Rifampin) [74], 2a6h (Streptolydigin) [75], 4yfx (Myxopyronin B) [76], 6fbv (Fidaxomicin) [77], 6n60 (Microcin J25) [78], 4mex (Salinamide A) [79]. S. cerevisiae RNAPII was obtained from pdb code 3cqz [80]. For clarity, only one compound for target site is shown.