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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 SP? 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 SP? 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.

SUBMITTER: Forrest D

PROVIDER: S-EPMC5467207 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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

Forrest David D James Katherine K Yuzenkova Yulia Y Zenkin Nikolay N

Nature communications 20170606

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 abse ...[more]

PMID: 28585540

Similar Datasets

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

Project description:Transcription, the copying of genetic information into RNA, is accomplished by multi-subunit RNA polymerases (msRNAPs) in all living organisms. msRNAPs are highly conserved in evolution and invariantly share a ~400 kDa five-subunit catalytic core1,2. A group of hypothetical single-chain proteins of unknown function, present in various bacteria and bacteriophages, was predicted to be distantly related to msRNAPs, having diverged from them before the Last Universal Common Ancestor3. Here, we studied a ~100 kDa protein, YonO, from this group encoded by the bacteriophage SPβ of Bacillus subtilis. We show that despite homology to only several amino acids of msRNAP, and the absence of most of the conserved domains, YonO is a highly processive and fast DNA-dependent RNA polymerase. Unlike msRNAPs, YonO can start specific transcription on double-stranded DNA without additional factors. We show that YonO is a bona fide RNAP of SPβ bacteriophage that transcribes its late genes. This new class of RNAPs may represent an intermediate step in the evolution of an ancestor of all msRNAPs.

2016-09-30 | GSE80786 | GEO

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Assembly of a dsRNA synthesizing complex: RNA-DEPENDENT RNA POLYMERASE 2 contacts the largest subunit of NUCLEAR RNA POLYMERASE IV.

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Transcription initiation in a single-subunit RNA polymerase proceeds through DNA scrunching and rotation of the N-terminal subdomains.

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The phage N4 virion RNA polymerase catalytic domain is related to single-subunit RNA polymerases.

Project description:In vitro, bacteriophage N4 virion RNA polymerase (vRNAP) recognizes in vivo sites of transcription initiation on single-stranded templates. N4 vRNAP promoters are comprised of a hairpin structure and conserved sequences. Here, we show that vRNAP consists of a single 3500 amino acid polypeptide, and we define and characterize a transcriptionally active 1106 amino acid domain (mini-vRNAP). Biochemical and genetic characterization of this domain indicates that, despite its peculiar promoter specificity and lack of extensive sequence similarity to other DNA-dependent RNA polymerases, mini-vRNAP is related to the family of T7-like RNA polymerases.

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Project description:Transcription termination is one of the least understood processes of gene expression. As the prototype model for transcription studies, the single-subunit T7 RNA polymerase (RNAP) is known to respond to two types of termination signals, but the mechanism underlying such termination, especially the specific elements of the polymerase involved, is still unclear, due to a lack of knowledge with respect to the structure of the termination complex. Here we applied phage-assisted continuous evolution to obtain variants of T7 RNAP that can bypass the typical class I T7 terminator with stem-loop structure. Through in vivo selection and in vitro characterization, we discovered a single mutation (S43Y) that significantly decreased the termination efficiency of T7 RNAP at all transcription terminators tested. Coincidently, the S43Y mutation almost eliminates the RNA-dependent RNAP (RdRp) activity of T7 RNAP without impeding the major DNA-dependent RNAP (DdRp) activity of the enzyme. S43 is located in a hinge region and regulates the transformation between transcription initiation and elongation of T7 RNAP. Steady-state kinetics analysis and an RNA binding assay indicate that the S43Y mutation increases the transcription efficiency while weakening RNA binding of the enzyme. As an enzymatic reagent for in vitro transcription, the T7 RNAP S43Y mutant reduces the undesired termination in run-off RNA synthesis and produces RNA with higher terminal homogeneity.

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