Structural basis of RNA polymerase inhibition by viral and host factors

Simona Pilotto,Luis Miguel Díaz-Santín,Finn Werner,Alan C M Cheung,Thomas Fouqueau,Dorota Matelska,Soizick Lucas-Staat,Carol Sheppard,Natalya Lukoyanova,David Prangishvili

doi:10.1038/s41467-021-25666-5

Abstract

RNA polymerase inhibition plays an important role in the regulation of transcription in response to environmental changes and in the virus-host relationship. Here we present the high-resolution structures of two such RNAP-inhibitor complexes that provide the structural bases underlying RNAP inhibition in archaea. The Acidianus two-tailed virus encodes the RIP factor that binds inside the DNA-binding channel of RNAP, inhibiting transcription by occlusion of binding sites for nucleic acid and the transcription initiation factor TFB. Infection with the Sulfolobus Turreted Icosahedral Virus induces the expression of the host factor TFS4, which binds in the RNAP funnel similarly to eukaryotic transcript cleavage factors. However, TFS4 allosterically induces a widening of the DNA-binding channel which disrupts trigger loop and bridge helix motifs. Importantly, the conformational changes induced by TFS4 are closely related to inactivated states of RNAP in other domains of life indicating a deep evolutionary conservation of allosteric RNAP inhibition.

Highlights

RNA polymerase inhibition plays an important role in the regulation of transcription in response to environmental changes and in the virus-host relationship
As similar structural perturbations occur in other inhibited states of RNA polymerases (RNAPs), our results demonstrate that the allosteric inhibition of the RNA polymerase is evolutionary conserved across all domains of life
The Sulfolobus acidocaldarius (Saci) RNAP structure includes eight conserved metal centres, six zinc coordinating motifs, a redox-inactive[18] cubane [3Fe-4S] iron-sulphur cluster, as well as the catalytic magnesium ion (MgA) that is characteristic for multisubunit, double-psi beta barrel (DPBB) RNAPs (Fig. 1b and Supplementary Figs. 2, 3, and 4a)

Summary

Introduction

RNA polymerase inhibition plays an important role in the regulation of transcription in response to environmental changes and in the virus-host relationship. The host encoded and constitutively expressed Gfh[1] (Gre-factor homologue-1)[8] and DksA (DnaK suppressor A)[9] are inhibitors of bacterial RNAP that enable the fine-tuning of transcription, and belong to the group of factors that act through the nucleotide triphosphates (NTPs) entry funnel of RNAP, or secondary channel[10] These include positive regulators such as the eukaryotic RNAPII TFIIS11, archaeal TFS12,13 and bacterial GreA/B14,15 that enhance elongation and resolve stalled and backtracked transcription elongation complexes (TEC) by transcript cleavage. Rather than interfering with the binding of transcription factors or nucleic acids, TFS4 inhibits RNAP by inducing largescale conformational changes that result in the opening of the DNA-binding channel, and the disruption of active site motifs including the bridge helix and the trigger loop. As similar structural perturbations occur in other inhibited states of RNAP, our results demonstrate that the allosteric inhibition of the RNA polymerase is evolutionary conserved across all domains of life

Methods

Results

Conclusion