Structural basis of the complete poxvirus transcription initiation process.

Clemens Grimm,Aladar A Szalay,Julia Bartuli,Bettina Boettcher,Utz Fischer

doi:10.1038/s41594-021-00655-w

Abstract

Poxviruses express their genes in the cytoplasm of infected cells using a virus-encoded multi-subunit polymerase (vRNAP) and unique transcription factors. We present cryo-EM structures that uncover the complete transcription initiation phase of the poxvirus vaccinia. In the pre-initiation complex, the heterodimeric early transcription factor VETFs/l adopts an arc-like shape spanning the polymerase cleft and anchoring upstream and downstream promoter elements. VETFI emerges as a TBP-like protein that inserts asymmetrically into the DNA major groove, triggers DNA melting, ensures promoter recognition and enforces transcription directionality. The helicase VETFs fosters promoter melting and the phospho-peptide domain (PPD) of vRNAP subunit Rpo30 enables transcription initiation. An unprecedented upstream promoter scrunching mechanism assisted by the helicase NPH-I probably fosters promoter escape and transition into elongation. Our structures shed light on unique mechanisms of poxviral gene expression and aid the understanding of thus far unexplained universal principles in transcription.

Highlights

Poxviruses express their genes in the cytoplasm of infected cells using a virus-encoded multi-subunit polymerase and unique transcription factors
We used complete virus-encoded multi-subunit RNA polymerase (vRNAP) purified from HeLa cells infected with an engineered vaccinia strain to reconstitute FLAG-tagged[21] vRNAP pre-initiation and initially transcribing vaccinia vRNAP complexes
Active complete vRNAP was used to reconstitute transcription complexes on a vaccinia early promoter scaffold consisting of the critical region (CR), a non-complementary bubble including the transcription start site (TSS) (+1) and a template cassette lacking G nucleotides (Fig. 1a)

Summary

Introduction

Poxviruses express their genes in the cytoplasm of infected cells using a virus-encoded multi-subunit polymerase (vRNAP) and unique transcription factors. Eukarya use three structurally related nuclear multi-subunit RNAPs (Pol I, Pol II and Pol III) to transcribe distinct subsets of genes[1] They cooperate with distinct sets of transcription factors (TFs) that enable recruitment to specific classes of promoters. The poxviruses, which cause smallpox in humans and various zoonoses, are a remarkable exception[12,13,14] They multiply exclusively in the cytoplasm of infected cells and depend on their own set of factors ensuring gene expression and replication. Studies on the prototypical vaccinia virus identified a virus-encoded multi-subunit RNA polymerase (vRNAP) associated with factors that ensure the production of polyadenylated and m7G-capped mRNA15–19. The structural analysis of these complexes uncovers principles of promoter recognition and melting, template strand capture and an unprecedented helicase-assisted upstream DNA scrunching mechanism prior to promoter escape. Our study unravels the mechanisms of poxviral gene expression and helps to understand far unexplained universal principles of transcription

Methods

Results

Conclusion