Single-peptide DNA-dependent RNA polymerase homologous to multi-subunit RNA polymerase

Abstract

Transcription in all living organisms is accomplished by multi-subunit RNA polymerases (msRNAPs). msRNAPs are highly conserved in evolution and invariably share a ∼400 kDa five-subunit catalytic core. Here we characterize a hypothetical ∼100 kDa single-chain protein, YonO, encoded by the SPβ prophage of Bacillus subtilis. YonO shares very distant homology with msRNAPs, but no homology with single-subunit polymerases. We show that despite homology to only a few amino acids of msRNAP, and the absence of most of the conserved domains, YonO is a highly processive DNA-dependent RNA polymerase. We demonstrate that YonO is a bona fide RNAP of the SPβ bacteriophage that specifically transcribes its late genes, and thus represents a novel type of bacteriophage RNAPs. YonO and related proteins present in various bacteria and bacteriophages have diverged from msRNAPs before the Last Universal Common Ancestor, and, thus, may resemble the single-subunit ancestor of all msRNAPs.

Figure 1. YonO is an RNAP.

(a) Detectible homology of YonO and msRNAP is marked in the alignment (identical amino acids—black, conserved substitutions—bold). Identical amino acids are shown on the crystal structure of E. coli msRNAP (pdbID 4IGC) as spheres. (b) YonO forms stable active elongation complexes. Scheme of the experiment and the sequences of nucleic acids used are shown next to the gel (here and after, for sequences, see Supplementary Table 2). Partial destruction of ECs upon addition of access of the non-template DNA strand (lane 5) is also commonly observed for msRNAP. (c) RNA extension in ECs (as in panel (b)) formed by wild-type YonO and mutant YonO carrying asparagine substitutions of the aspartate triad homologous to the absolutely conserved catalytic aspartate triad of msRNAPs. (d) Specificity of YonO and msRNAP to RNA versus DNA as primers and templates (see Supplementary Table 2 for sequences), and NTP versus dNTP as substrates. A higher molecular weight band in the third panel that coincides with the extension product is a contaminant in the preparation of the DNA primer. (e) Kinetics of DNA-dependent RNA polymerization by YonO and msRNAP in the presence of all NTPs. RNA in the EC was labelled at the 3′ end by incorporation of α-[³²P]GMP, shown in bold in the scheme next to the gel. (f) YonO is more error prone than msRNAPs. Kinetics of misincorporation by YonO and E. coli msRNAP. RNA was labelled as in panel (e). Note that RNAs of different sequences are well resolved in this Urea-PAGE excluding the possibility that the extension is caused by the contamination with correct NTPs. (g) Kinetics of RNA hydrolysis by YonO and msRNAP (see also Supplementary Fig. 1f).

Figure 2. YonO is a bona fide RNAP of SPβ.

(a) Western blot analysis of YonO expression upon SPβ induction with mitomycin C. DivIVA, a constitutively expressed cell division protein, was probed as a loading control. (b) Plaque formation assay with SPβ sensitive strain (CU1065) infected with lysates of mitomycin C (MitC)-induced wild type, ΔSPβ and ΔyonO B. subtilis strains. Parts of agar plates are shown. (c) A volcano plot of the gene expression fold change against P-value for the 4314 genes between WT strains with and without mitomycin C treatment. Genes were considered significant at a fold change of 2 and a P-value threshold of 0.05, following correction using the Benjamini–Hochberg false discovery rate. SPβ genes with statistically significant changes in expression are shown in green (N=157) with other significant genes in pink (N=1,072). Blue indicates all the genes (out of 4,314) without a statistically significant change in expression. (d) A volcano plot of the gene expression fold change against P-value for the 4,314 genes between wild-type and ΔyonO strains upon prophage induction. Criteria for significant change are as in panel c. Out of the 148 SPβ prophage genes induced by mitomycin C (see panel c), 37 were not expressed in the ΔyonO strain (pink dots with labels; see also Supplementary Fig. 2). Blue dots indicate all the genes (out of 4,314) without a statistically significant change in expression. (e) Confirmation of the transcription start site (TSS) of the promoter used by YonO (P_YONO) to transcribe the operon of SPβ late genes, as determined by RNA-seq, using primer extension. (f) Deletion of YonO does not affect transcription from constitutive P_VEG promoter of B. subtilis as confirmed by primer extension.