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.

痘病毒使用病毒编码的多亚基聚合酶 (vRNAP) 和独特的转录因子在受感染细胞的细胞质中表达其基因。我们展示了揭示痘病毒痘苗病毒完整转录起始阶段的冷冻电镜结构。在预启动复合物中，异二聚体早期转录因子 VETFs/l 采用弧形跨越聚合酶裂隙并锚定上游和下游启动子元件。VETFI 作为一种类似 TBP 的蛋白质出现，它不对称地插入 DNA 大沟，触发 DNA 熔化，确保启动子识别并加强转录方向性。解旋酶 VETF 促进启动子解链，而 vRNAP 亚基 Rpo30 的磷酸肽结构域 (PPD) 可启动转录。由解旋酶 NPH-I 辅助的前所未有的上游启动子清洗机制可能促进启动子逃逸和过渡到延伸。我们的结构揭示了痘病毒基因表达的独特机制，并有助于理解迄今为止无法解释的转录普遍原则。