A non-canonical multisubunit RNA polymerase encoded by a giant bacteriophage

Abstract

The infection of Pseudomonas aeruginosa by the giant bacteriophage phiKZ is resistant to host RNA polymerase (RNAP) inhibitor rifampicin. phiKZ encodes two sets of polypeptides that are distantly related to fragments of the two largest subunits of cellular multisubunit RNAPs. Polypeptides of one set are encoded by middle phage genes and are found in the phiKZ virions. Polypeptides of the second set are encoded by early phage genes and are absent from virions. Here, we report isolation of a five-subunit RNAP from phiKZ-infected cells. Four subunits of this enzyme are cellular RNAP subunits homologs of the non-virion set; the fifth subunit is a protein of unknown function. In vitro, this complex initiates transcription from late phiKZ promoters in rifampicin-resistant manner. Thus, this enzyme is a non-virion phiKZ RNAP responsible for transcription of late phage genes. The phiKZ RNAP lacks identifiable assembly and promoter specificity subunits/factors characteristic for eukaryal, archaeal and bacterial RNAPs and thus provides a unique model for comparative analysis of the mechanism, regulation and evolution of this important class of enzymes.

Figure 1.

Purification of phiKZ-encoded nvRNAP. (A) The sequence of steps used to purify nvRNAP is shown. (B) А Coomassie-stained SDS gel of fractions containing gp55 and gp74 during nvRNAP purification with the gel lane numbers corresponding to the numbers of steps from panel А. (C) Native PAGE analysis of nvRNAP from the final MonoQ purification step (upper panel) and subunit composition of the native PAGE band (lower panel). Individual protein bands were identified by mass-spectrometry.

Figure 2.

Conserved sequence and structural regions of the phiKZ nvRNAP subunits. The second largest (β in bacteria) and the largest (β’ in bacteria) cellular multisubunit RNAPs subunits are shown as arrows in panels A and B, respectively. Evolutionarily conserved sequence regions are labeled according to the Lane and Darst nomenclature: a1-a16 for β and a1-a20 for β’; the size of subunits and the positions of conserved regions are shown for Thermus thermophilus RNAP as a representative bacterial enzyme. Below the β and β’ subunits schemes ihe phiKZ nvRNAP subunits are shown, with sequence regions similar to corresponding msRNAP subunits identified and connected to each other by thin dotted lines. The alignments of conserved regions are presented in Supplementary Figure S1. The DPBB domains that form msRNAP catalytic centers are marked as ovals.

Figure 3.

In vitro transcription by phiKZ nvRNAP. (A) Multi-round run-off in vitro transcription by nvRNAP from PCR fragments containing an early (P_54E), middle (P_62M) and late (P_119L) phiKZ promoters. RO – run-off RNA products. (B) Primer extension mapping of 5′-ends of in vivo (left panel) and in vitro (right panel) transcripts from the phiKZ late promoter P_119L. DNA sequencing reactions with the same end-labeled primer used as sequence markers are indicated. The arrows indicate primer extension products. (C) In vitro abortive initiation reactions by nvRNAP and host P. aeruginosa RNAP σ⁷⁰-holoenzyme (Pa RNAP) from phiKZ late promoter (P_119L) and bacterial RNAP promoter T7 A1. 3 nt – trinucleotide abortive transcripts. (D) In vitro run-off transcription by nvRNAP and P. aeruginosa RNAP from phiKZ P_119L and T7 A1 promoters in the presence or absence of rifampicin. RO – run-off RNA products.

Figure 4.

Promoter specificity determinants of nvRNAP. (A) Sequence logos of phiKZ middle and late promoters. (B) Analysis of the phiKZ late promoter consensus motif 5′-TATG-3′ by point mutations. Left panel – in vitro run-off transcription by nvRNAP from late promoter P_119L and its derivatives. RO – run-off RNA products. Right panel – an alignment of nucleotide sequences of wild-type and mutant promoters (introduced substitutions are shown in bold typeface). The +1 start site is underlined. (C) Mutational analysis of upstream and downstream sequences of phiKZ late promoter P_119L. Left panel – in vitro run-off transcription by nvRNAP from chimeric templates based on phiKZ P_78M middle and P_119L late promoters. RO – run-off RNA products. Right panel – schematic representation of hybrid promoters used. The P_119L sequence is shown by a thin line; the P_78M is shown by a thick line. Numbers above the scheme indicate upstream (position −53 with respect to the start site) and downstream (position +51) boundaries of promoter fragments used. The late consensus TATG motif with the +1 start of transcription is underlined. The putative middle promoter conserved motif 5′-AAAATTACC-3′ is also indicated. The numbers at the right indicate the transcription activities relative to the positive control (wild-type P_119L). Average values and standard deviations from three independent experiments are presented.