Distinct pathways of RNA polymerase regulation by a phage-encoded factor

Abstract

Transcription antitermination is a common strategy of gene expression regulation, but only a few transcription antitermination factors have been studied in detail. Here, we dissect the transcription antitermination mechanism of Xanthomonas oryzae virus Xp10 protein p7, which binds host RNA polymerase (RNAP) and regulates both transcription initiation and termination. We show that p7 suppresses intrinsic termination by decreasing RNAP pausing and increasing the transcription complex stability, in cooperation with host-encoded factor NusA. Uniquely, the antitermination activity of p7 depends on the ω subunit of the RNAP core and is modulated by ppGpp. In contrast, the inhibition of transcription initiation by p7 does not require ω but depends on other RNAP sites. Our results suggest that p7, a bifunctional transcription factor, uses distinct mechanisms to control different steps of transcription. We propose that regulatory functions of the ω subunit revealed by our analysis may extend to its homologs in eukaryotic RNAPs.RNA polymerase | transcription antitermination | transcription pausing | omega subunit | NusA T ranscription promoters and terminators are the main "punctuation marks" that control the expression of individual genes in the genome. In bacteria, transcription termination is an essential regulatory step that determines the relative levels of expression of promoter-proximal and distal genes within operons. Depending on the factors involved, transcription termination in bacteria can be classified as intrinsic (depends on RNAencoded signals) or factor-dependent (requires additional protein factors such as Rho helicase). Intrinsic terminators consist of a G/C-rich hairpin formed in the nascent RNA followed by a downstream oligo(U) tract. During intrinsic termination, RNA polymerase (RNAP) pauses after synthesizing an oligo(U) sequence, accompanied by hairpin formation, leading to subsequent dissociation of the transcription elongation complex (TEC) (1, 2). Despite the relatively simple overall scenario, the exact mechanisms of pausing and hairpin-induced TEC destabilization remain an issue of debate (1, 3).Transcription antitermination is a widespread phenomenon involved in regulation of bacterial and phage operons (2). Transcription antitermination is often mediated by specialized protein factors that target host RNAP and can suppress termination at multiple sites in the operon. The well-studied examples of phage antiterminators include proteins N and Q of Escherichia coli phage λ and the gp39 protein of Thermus thermophilus phage P23-45. These factors suppress RNAP pausing, thus inhibiting the first step of the termination pathway (4-6). The antitermination activity of N and Q, but not gp39, is enhanced by cell-encoded proteins, including transcription elongation factor NusA, which by itself stimulates termination but reverses its activity to additionally stabilize the TEC in cooperation with N and Q (5, 6). Curiously, the N, Q, gp39, and NusA proteins all target the flexible β flap domain of RNAP (2, 4...

Gfh factors and NusA cooperate to stimulate transcriptional pausing and termination

“…NusA was previously shown to enhance pausing and intrinsic termination in various bacteria, including E. coli , Bacillus subtilis , T. thermophilus , and Xanthomonas oryzae . We demonstrated that Dra NusA also stimulates hairpin‐dependent pausing and intrinsic termination by Dra RNAP.…”

“…While the functions of some pauses are well established (synchronization of transcription with translation for hisP and, probably, consP), others may appear as an unavoidable consequence of variations in the transcribed DNA sequence. RNAP pausing is also the first step in the transcription termination pathway, and intrinsic terminators share common structural motifs with hairpin‐dependent pause signals, which may cause similar rearrangements of the TEC during termination .…”

“…The activity of NusA greatly depends on the secondary RNA structure, with the strongest effects observed for hairpin‐dependent pause signals . At the same time, NusA enhances the antipausing activities of several known transcription antiterminators, including the Q and N proteins of phage λ (reviewed in ) and p7 protein of phage Xp10 .…”

Regulation of transcriptional pausing through the secondary channel of RNA polymerase

“…In particular, both Gfh1 and Gfh2 strongly enhance recognition of two common types of the pausing signals found in bacteria, consensus and hairpin-dependent pauses, by Dra RNAP. They also stimulate intrinsic termination, which likely proceeds through the same structural intermediate(s) as hairpin-dependent pausing (9,35,36). Previously, the pause RNA hairpin was shown to induce long-range conformational changes of the TEC, including opening of the clamp domain and inhibition of the TL folding (12,13).…”

Mutations in the CRE pocket of bacterial RNA polymerase affect multiple steps of transcription

“…T7A1 promoter followed by λ tR2 terminator was obtained as in ( 20 ). DNA template containing a fragment of the E. coli rpoB gene fused to the λ P R promoter for measurements of elongation rates was described previously ( 21 ). DNA templates for analysis of hisP pausing were obtained by PCR from plasmid pIA226 containing λ P R promoter followed by the hisP site.…”