Structure and function of virion RNA polymerase of crAss-like phage (original) (raw)
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Host RNA polymerase inhibitors encoded by ϕKMV-like phages of pseudomonas
Virology, 2013
Pseudomonas RNA polymerase RNA polymerase inhibitor a b s t r a c t Escherichia coli bacteriophage T7 is a founding member of a large clade of podoviruses encoding a single-subunit RNA polymerase (RNAP). Phages of the family rely on host RNAP for transcription of early viral genes; viral RNAP transcribes non-early viral genes. T7 and its close relatives encode an inhibitor of host RNAP, the gp2 protein. Gp2 is essential for phage development and ensures that host RNAP does not interfere with viral RNAP transcription at late stages of infection. Here, we identify host RNAP inhibitors encoded by a subset of T7 clade phages related to fKMV phage of Pseudomonas aeruginosa. We demonstrate that these proteins are functionally identical to T7 gp2 in vivo and in vitro. The ability of some Pseudomonas phage gp2-like proteins to inhibit RNAP is modulated by N-terminal domains, which are absent from the T7 phage homolog. This finding indicates that Pseudomonas phages may use external or internal cues to initiate inhibition of host RNAP transcription and that gp2-like proteins from these phages may be receptors of these cues.
A non-canonical multisubunit RNA polymerase encoded by a giant bacteriophage
Nucleic Acids Research, 2015
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 rifampicinresistant 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.
Distinct pathways of RNA polymerase regulation by a phage-encoded factor
Proceedings of the National Academy of Sciences of the United States of America, 2015
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...
Journal of Biological Chemistry, 2007
Studies of the RNA-dependent RNA polymerase (RdRp) from poliovirus (PV), 3Dpol, have shown that Asn-297 permits this enzyme to distinguish ribose from 2-deoxyribose. All animal RNA viruses have Asn at the structurally homologous position of their polymerases, suggesting a conserved function for this residue. However, all prokaryotic RNA viruses have Glu at this position. In the presence of Mg 2؉ , the apparent affinity of Glu-297 3Dpol for 2-deoxyribonucleotides was decreased by 6-fold relative to wild type without a substantial difference in the fidelity of 2-dNMP incorporation. The fidelity of ribonucleotide misincorporation for Glu-297 3Dpol was reduced by 14-fold relative to wild type. A 4-to 11-fold reduction in the rate of ribonucleotide incorporation was observed. Glu-297 PV was unable to grow in HeLa cells due to a replication defect equivalent to that observed for a mutant PV encoding an inactive polymerase. Evaluation of the protein-(VPg)-primed initiation reaction showed that only half of the Glu-297 3Dpol initiation complexes were capable of producing VPg-pUpU product and that the overall yield of uridylylated VPg products was reduced by 20-fold relative to wild-type enzyme, a circumstance attributable to a reduced affinity for UTP. These studies identify the first RdRp derivative with a mutator phenotype and provide a mechanistic basis for the elevated mutation frequency of RNA phage relative to animal RNA viruses observed in culture. Although protein-primed initiation and RNA-primed elongation complexes employ the same polymerase active site, the functional differences reported here imply significant structural differences between these complexes.
Single-stranded RNA phages ToC
Single-Stranded RNA Phages: From Molecular Biology to Nanotechnology, 2020
This item is eligible for FREE Click and Collect without a minimum order subject to availability. Details This is a comprehensive guide to single-stranded RNA phages (family Leviviridae), first discovered in 1961. These phages played a unique role in early studies of molecular biology, the genetic code, translation, replication, suppression of mutations. Special attention is devoted to modern applications of the RNA phages and their products in nanotechnology, vaccinology, gene discovery, evolutionary and environmental studies. Included is an overview of the generation of novel vaccines, gene therapy vectors, drug delivery, and diagnostic tools exploring the role of RNA phage-derived products in the revolutionary progress of the protein tethering and bioimaging protocols. Key Features Presents the first full guide to single-stranded RNA phages Reviews the history of molecular biology summarizing the role RNA phages in the development of the life sciences Demonstrates how RNA phage-derived products have resulted in nanotechnological applications Presents an up-to-date account of the role played by RNA phages in evolutionary and environmental studies
Analysis of RNA phage fr coat protein assembly by insertion, deletion and substitution mutagenesis
Protein Engineering Design & Selection, 1993
A structure-function analysis of the icosahedral RNA bacteriophage/r coat protein (CP) assembly was undertaken using linker-insertion, deletion and substitution mutagenesis. Mutations were specifically introduced into either pre-existing or artificially created restriction enzyme sites within fr CP gene expressed in Escherichia coli from a recombinant plasmid. This directs synthesis of wild type protein that undergoes self-assembly and forms capsid-like particles indistinguishable morphologically and immunologically from native phage particles. A series of fr CP variants containing sequence alterations in the regions which are (i) exposed on the external surface of capsid or (ii) located on the contacting areas between CP subunits were obtained and their assembly properties investigated. The majority of mutants demonstrated reduction of assembly ability and formed either CP dimers (mutations at residues 2, 10, 63 or 129) or both dimer and capsid structures (residue 2 or 69). The exceptions were variants demonstrating normal assembly and containing insertions at residues 2, 50 or 129 of thefr CP. A third type of assembled structure was formed by a variant with a single amino acid substitution I104T. The aA-helix region (residues 97-111) is particularly sensitive to mutation and any alteration in this region decreases accumulation of mutant protein in E.coli. The relative contributions of particular fr CP domains in maintenance of capsid structural integrity as well as the possible capsid assembly mechanism are discussed.
Nucleic acids research, 2017
Infection of Escherichia coli by the T7 phage leads to rapid and selective inhibition of the bacterial RNA polymerase (RNAP) by the 7 kDa T7 protein Gp2. We describe the identification and functional and structural characterisation of a novel 7 kDa T7 protein, Gp5.7, which adopts a winged helix-turn-helix-like structure and specifically represses transcription initiation from host RNAP-dependent promoters on the phage genome via a mechanism that involves interaction with DNA and the bacterial RNAP. Whereas Gp2 is indispensable for T7 growth in E. coli, we show that Gp5.7 is required for optimal infection outcome. Our findings provide novel insights into how phages fine-tune the activity of the host transcription machinery to ensure both successful and efficient phage progeny development.
Mutilation of RNA phage Qβ virus-like particles: from icosahedrons to rods
Febs Letters, 2000
Icosahedral virus-like particles (VLPs) of RNA phage QL L are stabilized by four disulfide bonds of cysteine residues 74 and 80 within the loop between L L-strands F and G (FG loop) of the monomeric subunits, which determine the five-fold and quasisix-fold symmetry contacts of the VLPs. In order to reduce the stability of QL L VLPs, we mutationally converted the amino acid stretch 76-ANGSCD-81 within the FG loop into the 76-VGGVEL-81 sequence. It led to production in Escherichia coli cells of aberrant rod-like QL L VLPs, along with normal icosahedral capsids. The length of the rod-like particles exceeded 4^30 times the diameter of icosahedral QL L VLPs. ß
Rapid Mutagenesis and Purification of Phage RNA Polymerases
Protein Expression and Purification, 1997
based expression systems that allow rapid mutagenesis We have developed plasmid-based expression sys-and purification of T7 and other phage RNA polymertems that encode modified forms of T7 RNA polymer-ases. ase (RNAP) having 6-12 histidine residues fused to the Previous methods for purification of T7 RNAP have amino terminus. The histidine-tagged RNAPs (His-T7 included selective precipitation with salt, size-exclu-RNAPS) are indistinguishable from the wild-type (WT) sion chromatography, chromatography on ion exchange enzyme in nearly all biochemical assays. Similar plascolumns, and affinity chromatography on columns such mids that encode His-tagged T3 and SP6 RNAPs have as Affi-Gel blue or GTP-agarose (1-9). In general, also been constructed. To facilitate site-directed mutathese methods are time consuming and require dedigenesis of the RNAP gene, the size of the target plascated columns and chromatography equipment for each mid was minimized by using T7 RNAP itself as a seenzyme preparation. We were interested in rapid methlectable marker. BL21 (DCAT4) cells (which carry a ods for simultaneous purification of multiple samples chromosomal copy of the chloramphenicol acetylof RNAP, without the need for dialysis or de-salting transferase cat gene under control of a T7 promoter) between chromatography steps. We have developed are resistant to chloramphenicol when functional T7 two such methods, one for purification of conventional RNAP is expressed, thus allowing the selection and (unmodified) RNAPs, the other for RNAPs that have maintenance of the target plasmid in these cells. Mutabeen modified to include a histidine tag at their amino genesis is accomplished by denaturing the plasmid, termini. Both methods are rapid and give enzymes of annealing mutagenic DNA primers, and repairing the high quality which are suitable for use in transcription plasmid with T4 DNA polymerase. Two DNA primers assays and biochemical characterizations. are used: one corrects a defect in the bla gene, the A number of methods have been devised for engiother introduces the desired mutation into the RNAP neering proteins with affinity tags and the subsequent gene; 30-85% of the ampicillin-resistant transformants
The Journal of general virology, 2010
Chandipura virus (CHPV) is an emerging human pathogen associated with acute encephalitis and is related closely to vesicular stomatitis virus (VSV), a prototype rhabdovirus. Here, we demonstrate that the RNA polymerase L protein of CHPV exhibits a VSV-like RNA:GDP polyribonucleotidyltransferase (PRNTase) activity, which transfers the 5'-monophosphorylated (p-) viral mRNA start sequence to GDP to produce a capped RNA, and that the conserved HR motif in the CHPV L protein is essential for the PRNTase activity. Interestingly, the CHPV L protein was found to form two distinct SDS-resistant complexes with the CHPV mRNA and leader RNA start sequences; mutations in the HR motif significantly reduced the formation of the former complex (a putative covalent enzyme-pRNA intermediate in the PRNTase reaction), but not the latter complex. These results suggest that the rhabdoviral L proteins universally use the active-site HR motif for the PRNTase reaction at the step of the enzyme-pRNA inte...