Tail-tape-fused virion and non-virion RNA polymerases of a thermophilic virus with an extremely long tail

Abstract

Thermus thermophilus bacteriophage P23-45 encodes a giant 5,002-residue tail tape measure protein (TMP) that defines the length of its extraordinarily long tail. Here, we show that the N-terminal portion of P23-45 TMP is an unusual RNA polymerase (RNAP) homologous to cellular RNAPs. The TMP-fused virion RNAP transcribes pre-early phage genes, including a gene that encodes another, non-virion RNAP, that transcribes early and some middle phage genes. We report the crystal structures of both P23-45 RNAPs. The non-virion RNAP has a crab-claw-like architecture. By contrast, the virion RNAP adopts a unique flat structure without a clamp. Structure and sequence comparisons of the P23-45 RNAPs with other RNAPs suggest that, despite the extensive functional differences, the two P23-45 RNAPs originate from an ancient gene duplication in an ancestral phage. Our findings demonstrate striking adaptability of RNAPs that can be attained within a single virus species.

Fig. 1. Validation of P23-45 gp96 vRNAP and gp64 nvRNAP.

a Schematic of 5,002-residue tail tape measure protein gp96. The positions of membrane and metallopeptidase domains, the metal binding ⁷⁷⁹DFDGD⁷⁸³ sequence, and C-terminal boundaries of truncated gp96 variants used in this experiment are all indicated. b SDS PAGE of purified recombinant gp64 and gp96 variants. c, d Extension of a Cy5-labeled RNA primer within the RNA–DNA scaffold shown at the bottom in the presence of rNTPs by the truncated versions of gp96 (c) and by gp64 (d). Bands corresponding to the initial (10 nt) and extended (29 nt) RNA primer are labeled with one and two asterisks, correspondingly. Micro RNA marker was loaded on the same gels and visualized by staining with SYBR Gold. e Western blots of proteins from P23-45 virions with polyclonal antibodies against gp64 and against the 1050-residue fragment of gp96. For each panel shown, the assay was performed twice for each of two biological replicates. Uncropped gels can be found in Supplementary Fig. 1. Source data used in (b–e) are provided as a Source Data file.

Fig. 2. Transcription of the P23-45 genome by gp96 vRNAP and gp64 nvRNAP.

a Schematic of P23-45 genome with early (green), middle (violet) and late (blue) genes is shown at the top. Below, coverage tracks of DNA co-immunoprecipitated from T. thermophilus culture 5 min post-infection with P23-45 with antibodies raised against the 1540-residue fragment of gp96 and against gp64 are shown. The control track was obtained using an input DNA without co-immunoprecipitation. b ONT-cappable-seq results mapped on the P23-45 genome. Total RNA prepared from infected T. thermophilus cells collected 5 min post-infection was used. Transcription start and termination sites are shown below (Supplementary Data 1). c Conserved motifs present upstream of indicated groups of transcription start sites (TSSs) and transcription termination sites (TTSs) identified by MEME are shown. The motifs are framed with frame colors corresponding to colors of features in (b). Source data used in (c) are provided as a Source Data file.

Fig. 3. Structures of P23-45 gp96 vRNAP and gp64 nvRNAP.

a Overall structures of P23-45 gp96 vRNAP and gp64 nvRNAP are shown as ribbon models in two orientations and compared with the structures of T. thermophilus RNAP (PDB: 2O5J) and N. crassa QDE-1 (PDB: 2J7N). The conserved structural elements are colored (DPBB-A—orange; DPBB-B—blue; duplex-binding helix—brown; bridge helix—pink; trigger loop—green). Clamp domains are structurally variable (light blue) and the corresponding part in the gp96 structure (residues 613–679) is disordered. b The central regions of P23-45 gp96 vRNAP, gp64 nvRNAP, N. crassa QDE-1 (PDB: 2J7N), phi14:2 gp66 (PDB: 6VR4), and T. thermophilus RNAP (PDB: 2O5J) are shown in the same orientation. Local structures conserved in some RNAPs are highlighted: those conserved with gp96 but not with T. thermophilus RNAP—in yellow; those conserved with T. thermophilus RNAP but not with gp96 – in cyan; those conserved only between QDE-1 and gp66 – in purple. Other small elements conserved in all compared RNAPs are in lime.

Fig. 4. A model of the gp96 RNAP elongation complex.

The gp96 elongation complex was modeled by superimposing gp96 RNAP and the elongation complex of T. thermophilus RNAP (PDB: 2O5J). The same color scheme as in Fig. 3 is applied for gp96 RNAP. The nucleic acid models were copied from 2O5J (RNA: cyan; template DNA: red; non-template DNA: black).

Fig. 5. Relationships between RNAP families and functions of P23-45 RNAPs during infection.

a Sequence similarity dendrogram. UPGMA dendrogram was built using HHalign pairwise score matrix for 23 multiple alignments of distinct RNAP families. Alignments are provided in the Supplementary Data 3. *Branches within 3 distance units of the tree depth (see the scale under the tree) usually reliably reflect sequence similarity based on previous observations. Abbreviations: nvRNAP—non-virion RNAP, vRNAP—virion RNAP; QDE1—RNA/DNA-dependent RNA polymerase QDE-1. b Proposed mechanism of P23-45 pre-early genes transcription by gp96 vRNAP fused to tail tape measure protein (TMP) concomitantly with phage DNA translocation through the TMP pore. One of pre-early genes encodes gp64 nvRNAP, which transcribes all early and some middle genes. Late genes of the phage are transcribed by host RNAP (not shown). The figure was created using BioRender.com.