biostudies-literatureJungwirth SDeutsche Forschungsgemeinschaft (German Research Foundation)Volkswagen Foundation (VolkswagenStiftung)1648https://www.ebi.ac.uk/biostudies/studies/S-EPMC12917281biostudies-literatureUnknown17(1)The recruitment of RNA polymerase to gene promoters is a critical step in gene expression. For RNA polymerase II, this process is initiated by TBP and TFIIB, with homologs of these TBP/TFIIB pairs found in all known multi-subunit RNA polymerase systems. Here, we describe a mode of promoter recognition by the poxviral intermediate transcription factor 3, VITF-3. This heterodimeric factor comprises an atypical TBP/TFIIB pair forming a stable ring structure inert towards DNA in the absence of viral RNA polymerase. Promoter recognition instead requires concerted VITF-3 and viral RNA polymerase binding, as shown by cryo-EM analysis of the intermediate pre-initiation complex. During the formation of this complex, viral RNA polymerase facilitates ring opening and loading of VITF-3 onto the promotNature communicationsCooperative clamp-mediated promoter recognition by poxviral RNA polymerase and its TBP/TFIIB-like partner.PMC129172819B813Fi 573/27-1Fischer USchlosser ALamer SBartuli JGrimm CJungwirth SfalseCooperative clamp-mediated promoter recognition by poxviral RNA polymerase and its TBP/TFIIB-like partner.The recruitment of RNA polymerase to gene promoters is a critical step in gene expression. For RNA polymerase II, this process is initiated by TBP and TFIIB, with homologs of these TBP/TFIIB pairs found in all known multi-subunit RNA polymerase systems. Here, we describe a mode of promoter recognition by the poxviral intermediate transcription factor 3, VITF-3. This heterodimeric factor comprises an atypical TBP/TFIIB pair forming a stable ring structure inert towards DNA in the absence of viral RNA polymerase. Promoter recognition instead requires concerted VITF-3 and viral RNA polymerase binding, as shown by cryo-EM analysis of the intermediate pre-initiation complex. During the formation of this complex, viral RNA polymerase facilitates ring opening and loading of VITF-3 onto the promot2026-01-01T00:00:00Z2026 Feb2026-07-16T10:06:11.679Z2026-07-09T10:48:55.034ZS-EPMC129172814170860210.1038/s41467-026-69571-1