All cellular RNA polymerases (RNAPs) across Bacteria, Archaea, and Eukarya share a conserved catalytic core, yet bacterial and archaeal-eukaryotic RNAPs diverged after separation from the last universal common ancestor. This evolutionary split produced distinct subunit compositions and fundamentally different requirements for external factors during transcription initiation. Bacterial RNAP relies on a σ factor, whereas archaeal-eukaryotic RNAPs require a more extensive set of general transcription factors (GTFs) to bind promoter DNA, unwind the duplex, and position the template strand within the active site cleft. Notably, despite the close structural similarity between archaeal and eukaryotic RNAPs, the requirement for GTFs became further specialized after the emergence of Eukarya. This divergence raises the question of whether differences in intrinsic conformational flexibility and dynamics of these RNAPs contribute to distinct promoter-loading pathways. In this study, we addressed this question using cryo-electron microscopy (cryo-EM) to examine archaeal RNAPs from Euryarchaeota and Crenarchaeota alongside yeast RNAP II. Archaeal RNAP displays a highly dynamic DNA binding clamp domain that samples a broad spectrum of open and closed states, whereas RNAP II predominantly adopts a closed clamp state. Both archaeal and eukaryotic RNAPs can be found in stalk-bound and stalk-less forms. Comparative structural analyses further reveal a unique conformational transition in crenarchaeal RNAP associated with clamp opening. Together, these findings define the intrinsic clamp-conformational landscapes across the archaeal-eukaryotic lineage and suggest that evolutionary tuning of clamp flexibility and dynamics contributes to distinct GTF-dependent promoter-loading mechanisms.
George Nkansah Rost Fordjour, L. Palao, Kenji Murakami et al.· Journal of Biological Chemis...· 0 citations
ABSTRACT Coliphage N4 employs a unique infection and transcription strategy in which early gene expression is driven by a virion-encapsidated RNA polymerase (vRNAP) that is injected into the host cytoplasm upon infection. Despite extensive biochemical and crystallographic studies of the polymerase domain of vRNAP, the structural organization and regulatory roles of the N-terminal domain (NTD) and C-terminal domain (CTD) regions of the 3,500-residue-long whole enzyme have remained unresolved. Here, we report the cryo-electron microscopy (cryo-EM) structures of full-length N4 vRNAP in its apo state and in a transcription initiation complex (TIC) with promoter DNA and initiating nucleotides. The apo structure reveals a modular architecture in which an α-helical CTD packs against the Pol domain to stabilize an autoinhibited conformation characterized by occlusion of the nucleotide-binding site through tight contact between the plug module and motif B loop. In contrast, promoter binding induces conformational rearrangements that displace the motif B loop from the active site and separate the CTD from the Pol domain. The NTD is unresolved in both states, consistent with substantial intrinsic flexibility, and supporting its proposed role in membrane association and genome injection. Structural modeling suggests that domain segmentation and conformational plasticity may enable translocation of vRNAP through the ~30 Å wide phage tail channel during infection. Together, these results define the molecular architecture of full-length vRNAP and establish a structural framework for understanding how the conformational transition of vRNAP is coupled to its ejection, DNA injection, and early gene expression. IMPORTANCE This study investigates the structure of full-length bacteriophage N4 virion RNA polymerase (vRNAP), one of the largest known single-subunit RNA polymerases. The functions of its extensive N- and C-terminal regions remained unknown. Our work uncovers how the C-terminal domain regulates polymerase activity through a structural “switch” that locks the enzyme in an inactive state until it recognizes its promoter DNA. These findings explain how the phage prevents premature transcription and ensures precise control of early gene expression during infection. By integrating structures with the architecture of the N4 phage particle, we propose a mechanism by which this vRNAP is transported through the narrow phage tail into the host cell. Together, this work provides fundamental insight into phage transcription and viral gene regulation. This study investigates the structure of full-length bacteriophage N4 virion RNA polymerase (vRNAP), one of the largest known single-subunit RNA polymerases. The functions of its extensive N- and C-terminal regions remained unknown. Our work uncovers how the C-terminal domain regulates polymerase activity through a structural “switch” that locks the enzyme in an inactive state until it recognizes its promoter DNA. These findings explain how the phage prevents premature transcription and ensures precise control of early gene expression during infection. By integrating structures with the architecture of the N4 phage particle, we propose a mechanism by which this vRNAP is transported through the narrow phage tail into the host cell. Together, this work provides fundamental insight into phage transcription and viral gene regulation.
M. Narwal, Y. Shin, Katsuhiko S. Murakami· Journal of Bacteriology· 0 citations
