Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Coliphages”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,279 records · Page 71Linked to original sources
PubMed2005

Structure of lambda CII: implications for recognition of direct-repeat DNA by an unusual tetrameric organization.

The temperate coliphage lambda, after infecting its host bacterium Escherichia coli, can develop either along the lytic or the lysogenic pathway. Crucial to the lysis/lysogeny decision is the homotetrameric transcription-activator protein CII (4 x 11 kDa) of the phage that binds to a unique direct-repeat sequence T-T-G-C-N6-T-T-G-C at each of the three phage promoters it activates: p(E), p(I), and p(aQ). Several regions of CII have been identified for its various functions (DNA binding, oligomerization, and susceptibility to host protease), but the crystal structure of the protein long remained elusive. Here, we present the three-dimensional structure of CII at 2.6-angstroms resolution. The CII monomer is comprised of four alpha helices and a disordered C terminus. The first three helices (alpha1-alpha3) form a compact domain, whereas the fourth helix (alpha4) protrudes in different orientations in each subunit. A four-helix bundle, formed by alpha4 from each subunit, holds the tetramer. The quaternary structure can be described as a dimer of dimers, but the tetramer does not exhibit a closed symmetry. This unusual quaternary arrangement allows the placement of the helix-turn-helix motifs of two of the four CII subunits for interaction with successive major grooves of B-DNA, from one face of DNA. This structure provides a simple explanation for how a homotetrameric protein may recognize a direct-repeat DNA sequence rather than the inverted-repeat sequences of most prokaryotic activators.

Amino Acid Sequence↗
PubMed2003

Escherichia coli single-stranded DNA-binding protein mediates template recycling during transcription by bacteriophage N4 virion RNA polymerase.

Coliphage N4 virion RNA polymerase (vRNAP), the most distantly related member of the T7-like family of RNA polymerases, is responsible for transcription of the early genes of the linear double-stranded DNA phage genome. Escherichia coli single-stranded DNA-binding protein (EcoSSB) is required for N4 early transcription in vivo, as well as for in vitro transcription on super-coiled DNA templates containing vRNAP promoters. In contrast to other DNA-dependent RNA polymerases, vRNAP initiates transcription on single-stranded, promoter-containing templates with in vivo specificity; however, the RNA product is not displaced, thus limiting template usage to one round. We show that EcoSSB activates vRNAP transcription at limiting single-stranded template concentrations through template recycling. EcoSSB binds to the template and to the nascent transcript and prevents the formation of a transcriptionally inert RNA:DNA hybrid. Using C-terminally truncated EcoSSB mutant proteins, human mitochondrial SSB (Hsmt SSB), phage P1 SSB, and F episome-encoded SSB, as well as a Hsmt-EcoSSB chimera, we have mapped a determinant of template recycling to the C-terminal amino acids of EcoSSB. T7 RNAP contains an amino-terminal domain responsible for binding the RNA product as it exits from the enzyme. No sequence similarity to this domain exists in vRNAP. Hereby, we propose a unique role for EcoSSB: It functionally substitutes in N4 vRNAP for the N-terminal domain of T7 RNAP responsible for RNA binding.

Amino Acid Sequence↗
PubMed1975

Ribosomal protein S1 and polypeptide chain initiation in bacteria.

Among several subspecies of 30S subunits of Escherichia coli observed by polyacrylamide-agarose gel electrophoresis, only the slow-moving, protein S1-containing subspecies participates in the formation of the 30S initiation complex with coliphage MS2 RNA as mRNA; the other subspecies retain activity with AUG as mRNA; they are also active in the poly(U)-directed binding of Phe-tRNA. Protein S1 from Caulobacter crescentus substitutes for E. coli S1 despite the fact that C. crescentus ribosomes do not bind MS2 RNA. Under appropriate conditions, the entire population of E. coli 30S subunits can be isolated as the S1-containing subspecies. Protein S1 is lost by salt treatment of ribosomes.

Bacteria↗
PubMed1982

Carboxy terminus of polyoma middle-sized tumor antigen is required for attachment to membranes, associated protein kinase activities, and cell transformation.

We have constructed a transformation-defective polyoma virus mutant (Py 1387-T) that directs the synthesis of a normal small tumor antigen, a functional large tumor antigen, and a truncated (51,000-dalton) middle-sized tumor (mT) antigen that lacks 37 amino acids at its COOH terminus. The shortened mT polypeptide is missing the hydrophobic "tail" thought to be responsible for the anchorage of this protein into the plasma membrane and is in fact in cytosol fractions. This truncated mT polypeptide is inactive in an in vitro protein kinase assay and is altered in its phosphorylation in vivo. Mutant 1387-T differs from wild-type virus in having a T.A base pair instead of a C.G base at nucleotide position 1387. This change was introduced into viral DNA by using a synthetic undecanucleotide as a specific mutagen. Wild-type polyoma DNA was rendered single stranded by molecular cloning into coliphage M13. The oligonucleotide, which hybridizes with a mismatch at the site to be altered, was used to prime the synthesis of double-stranded closed circular DNA. Progeny recombinant phage were screened by DNA sequence analysis for the desired base change. The polyoma mutant was reconstructed from recombinant phage replicative form DNA molecules containing the mutation.

Animals↗
PubMed1982

The yeast his3 promoter contains at least two distinct elements.

Phenotypic analysis of 65 mutations indicates that the yeast his3 promoter is composed of at least two separate regions of DNA. Each is necessary, but neither is sufficient for wild-type levels of his3 expression. Deletion mutations that destroy either promoter element express his3 poorly or not at all. The upstream element is located between 112 and 155 base pairs before the site of transcriptional initiation (nucleotides -112 to -155). A comparison of derivatives strongly suggests that the downstream element maps somewhere between nucleotides -32 and -52 and includes a sequence between nucleotides -45 and -52. This location coincides with sequences conserved before most eukaryotic genes(the TATA box region). By using derivatives in which his3 sequences are replaced by a small fragment of coliphage M13 DNA, three properties of the his3 promoter were established. First, his3 TATA box deletions fail to express his3 because they lack specific sequences and not because they disrupt spacing relationships between other sequences. Second, the TATA box region can be replaced functionally by the one orientation of the M13 DNA fragment that contains a TATA-like sequence. Third, the distance between the two elements (normally 90 base pairs) can be varied between 40 and 160 base pairs without markedly affecting promoter function. These results strongly suggest that yeast RNA polymerase II, unlike its Escherichia coli counterpart, does not bind simultaneously to both promoter elements, and they add further support to the view that the upstream element is not part of a transcriptionally competent binding site. This ability of the his3 upstream promotor element to act at a long and variable distance is similar to properties of viral enhancer sequences and is reminiscent of position effects in yeast.

Base Sequence↗
PubMed1983

Developmental cell interactions in Myxococcus xanthus and the spoC locus.

The product(s) of the Myxococcus xanthus spoC locus is required for two multicellular activities in fruiting body development, rippling and sporulation. Ripples, which are formed early in development, are spatially separated ridges of cells that move synchronously. Myxospores are heat-resistant resting cells that are formed near the end of the developmental process. To investigate the function of spoC, it was cloned in an Escherichia coli plasmid, then transferred to M. xanthus by specialized transduction with coliphage P1. The plasmid, which cannot replicate in M. xanthus, integrated into the M. xanthus chromosome, producing two copies of the spoC locus in tandem. Cells containing one copy of a mutant allele and one copy of the wild-type allele displayed the wild-type phenotype. Cells containing two different mutant alleles failed to ripple or sporulate, implying that all four independent spoC mutations are in the same gene or unit of transcription. Homozygous mutant duplications arose from constructions in which DNA from a spo(+) donor was transduced into a spoC recipient, or vice versa, at an average frequency of 14%, indicating that gene conversion was a frequent event.

Journal Article↗
PubMed1984

Intervening sequence in the thymidylate synthase gene of bacteriophage T4.

The continuous sequence of 2.3 kilobases in a 3-kilobase DNA fragment encoding the structural gene for coliphage T4 thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) was determined by using the M13 dideoxy chain-termination method. From the coding information within this gene and that provided by sequence analysis of selected CNBr peptides from the protein product, the primary structure of T4 thymidylate synthase was determined. The most significant finding of these studies is the presence of a 1017-base-pair interruption two-thirds of the way through the nucleotide sequence of the structural gene. The 5'- and 3'-terminal ends of this intron are demarcated by an apparent stop and start codon, respectively. The corresponding methionine preceding the second coding region of the synthase is not incorporated into the final protein product. Structural evidence confirming the presence of the intervening sequence in the phage genome was obtained by restriction and hybridization analysis. Support for the presence of the intron was also obtained at the functional level by enzyme expression studies using selected td gene fragments. This work also confirms the findings of Purohit and Mathews [ Purohit , S. & Mathews , C. K. (1983) Fed. Proc. Fed. Am. Soc. Exp. Biol. 42, 1759], which reveal that the termination codon for the dihydrofolate reductase gene and the triplet initiating thymidylate synthase overlap by a four-base stretch, A-T-G-A. The implications of this unusual gene arrangement are discussed.

Amino Acid Sequence↗
PubMed1984

Pausing and premature termination of human RNA polymerase II during transcription of adenovirus in vivo and in vitro.

The major late transcriptional unit of adenovirus type 2 has served as a model for studying transcription in eukaryotes. We report that pausing and premature termination are intrinsic to the transcription of this transcriptional unit by RNA polymerase II. In vivo and in isolated nuclei, transcription pauses at discrete sites proximal to the initiation site and can prematurely terminate at nucleotide 175 and possibly also at nucleotide 120. The prematurely terminated RNAs are not associated with the transcription complexes and accumulate in the cell nucleus in vivo, whereas paused RNAs remain associated with the transcription complexes and elongate into full-length transcripts. Pausing is also reproduced in the transcription complexes in a soluble system. 5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole enhances pausing but not premature termination, and its action is reversible. The proposed premature termination site at nucleotide 175 in adenovirus type 2 bears sequence homology to the tR1 site in coliphage lambda.

Adenoviruses, Human↗
PubMed1986

The fusion-related hydrophobic domain of Sendai F protein can be moved through the cytoplasmic membrane of Escherichia coli.

Recent work on a prokaryotic membrane protein, gene III protein (pIII) of coliphage f1, showed that polypeptide segments of sufficient hydrophobicity functioned to stop transfer of the polypeptide across the cell membrane: strings of 16 or more hydrophobic amino acids sufficed. A fusion-related hydrophobic domain (FRHD) of Sendai F protein, a sequence of 26 consecutive uncharged residues, has been implicated in the fusion of the viral membrane envelope and the target-cell membrane through a hydrophobic interaction. As it is located on the exterior of the viral membrane, this sequence must be transferred across the host-cell membrane during synthesis. We have inserted either the FRHD or the F protein membrane anchor (the COOH-terminal region of the F protein) into an internal site of a secreted pIII, which lacks its natural membrane anchor. These two hydrophobic sequences behave in the bacteria just as they do in their natural eukaryotic cell host. The F protein membrane anchor functions to stop transfer, conferring a membrane-spanning topology to the F-pIII hybrid protein; however, the FRHD is moved through the cytoplasmic membrane and derivatives carrying this sequence are secreted to the periplasm. We discuss how the FRHD is compatible with passage through the membrane and yet is still able to mediate membrane fusion through a presumed hydrophobic interaction.

Amino Acid Sequence↗
PubMed1988

Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1.

The Cre protein encoded by the coliphage P1 is a 38-kDa protein that efficiently promotes both intra- and intermolecular synapsis and recombination of DNA both in Escherichia coli and in vitro. Recombination occurs at a specific site, called lox, and does not require any other protein factors. The Cre protein is shown here also to be able to cause synapsis of DNA and site-specific recombination in a mammalian cell line. A stable mouse cell line was established that expresses the Cre protein under the control of the Cd2+-inducible metallothionein I gene promoter. DNA recombination was monitored with DNA substrates containing two directly repeated lox sites. One such substrate is a circular plasmid with two directly repeated lox sites (lox2) flanking a marker gene and was introduced into cells by Ca3(PO4)2 transformation. As a second substrate we used a pseudorabies virus (a herpesvirus) containing a lox2 insertion designed to provide a sensitive detection system for recombination. In both cases, site-specific recombination in vivo is dependent on the presence of the Cre protein and occurs specifically at the 34-base-pair lox sites. These results demonstrate the controlled site-specific synapsis of DNA and recombination by a prokaryotic protein in mammalian cells and suggest that Cre-mediated site-specific recombination may be a useful tool for understanding and modulating genome rearrangements in eukaryotes.

Animals↗
PubMed1990

In vitro maturation and encapsidation of the DNA of transposable Mu-like phage D108.

Mu and D108 are related, temperate, transposable coliphages with unusual modes of DNA replication (transposition) and virion DNA maturation. These double-stranded DNA genomes replicate intrachromosomally and are matured and encapsidated linked to DNA sequences flanking the dispersed, integrated phage genomes. We have developed an in vitro system that employs crude lysates prepared from cells late in the Mu lytic cycle and that is proficient for both maturation and encapsidation of D108 DNA. Different forms of phage DNA were packaged at different efficiencies, with a circular pSC101::D108cts10 plasmid being most efficient, linearized plasmid less so, and mature virion DNA a poor substrate. The addition of purified D108 Ner protein to the reaction had no effect, whereas D108 repressor (c protein) inhibited the reaction. Escherichia coli integration host factor and D108 transposase proteins exerted an inhibitory effect on circular DNA substrates but had little effect on linear DNA packaging. This in vitro system, coupled with that developed for transposition, can now be used to biochemically dissect the protein and substrate requirements of these phages' DNA maturation pathway and the nature of the molecular switch between DNA transposition and encapsidation.

Bacteriophage mu↗
PubMed1992

Rational design of substituted tripyrrole peptides that complex with DNA by both selective minor-groove binding and electrostatic interaction with the phosphate backbone.

The structures of the compounds we call 3a, 3b, and 3c-compounds that incorporate (i) the tripyrrole peptide of the minor-groove-binding distamycin class of compounds and (ii) polyamine ligands that extend from the minor groove and can interact with phosphodiester bonds--were arrived at by computer-graphics designing by using the x-ray structure of distamycin A complexed in the minor groove of d(CGCAAATTTGCG)2. Compounds 3a, 3b, and 3c are elaborations of distamycin analog 2, designed for improved stability in solution and easier synthesis and purification, which itself binds weakly to DNA. Compounds 3a, 3b, and 3c have been synthesized, and the interaction of distamycin A, 2, 3a, 3b, and 3c with calf thymus DNA, poly(dA-dT), poly(dG-dC), poly(dI-dC), pBR322 superhelical plasmid DNA, and, in the case of 3b, T4 coliphage DNA have been studied. The following pertinent conclusions can be drawn. Binding of 3a, 3b, and 3c occurs in the minor groove of DNA and, because of favorable electrostatic interaction of diprotonated polyamine side chains and DNA phosphodiester linkages, the tenacity of DNA binding and site specificity of 3a, 3b, and 3c are comparable to that of native distamycin A. 3b has been found to induce changes in the superhelical density of pBR322 plasmid DNA. The study establishes that the central pyrrole N-CH3 substituent of 2 can be replaced by bulky polyamine metal ligands to create any number of compounds that bind into the minor groove at A + T-rich sites and are putative catalysts for the hydrolysis of DNA.

Animals↗
PubMed1993

A possible glycine radical in anaerobic ribonucleotide reductase from Escherichia coli: nucleotide sequence of the cloned nrdD gene.

During anaerobic growth of Escherichia coli an oxygen-sensitive ribonucleoside-triphosphate reductase, different from the aerobic ribonucleoside diphosphate-reductase (EC 1.17.4.1), produces the deoxyribonucleoside triphosphates required for DNA replication. The gene for the anaerobic enzyme has now been cloned and was found to contain a 2136-nucleotide coding region, corresponding to 712 amino acid residues, and an Fnr binding site 228 base pairs upstream of the initiator ATG. The deduced amino acid sequence shows 72% identity to a gene of coliphage T4, sunY, hitherto of unknown function, suggesting that the virus codes for its own anaerobic reductase. The location of an organic free radical formed during activation of the bacterial anaerobic reductase is proposed to be on Gly-681, since the pentapeptide RVCGY at positions 678-682 shows a striking similarity to the C-terminal sequence. RVSGY, of pyruvate formate-lyase. During activation of the anaerobically induced pyruvate formate-lyase, the glycine residue of the pentapeptide becomes an organic radical [Wagner, A. F. V., Frey, M., Neugebauer, F. A., Schäfer, W. & Knappe, J. (1992) Proc. Natl. Acad. Sci. USA 89, 996-1000]. The gene for the anaerobic reductase is located at a position around 96 min on the E. coli genomic map.

Amino Acid Sequence↗
PubMed1993

Gene 5.5 protein of bacteriophage T7 inhibits the nucleoid protein H-NS of Escherichia coli.

Gene 5.5 of coliphage T7 is one of the most highly expressed genes during T7 infection. Gene 5.5 protein, purified from cells overexpressing the cloned gene, purifies with the nucleoid protein H-NS of Escherichia coli during three chromatographic steps. A fusion protein of gene 5.5 protein and maltose binding protein also purifies with H-NS. The fusion protein binds to the DNA-H-NS complex and abolishes H-NS-mediated inhibition of transcription by Escherichia coli and T7 RNA polymerases in vitro. Expression of gene 5.5 also relieves the repression of the Escherichia coli proU promoter by H-NS in vivo. The change of leucine to proline at residue 30 of gene 5.5 protein abolishes the interaction between gene 5.5 protein and H-NS.

ATP-Binding Cassette Transporters↗
PubMed1997

RNA folding kinetics regulates translation of phage MS2 maturation gene.

The gene for the maturation protein of the single-stranded RNA coliphage MS2 is preceded by an untranslated leader of 130 nt, which folds into a cloverleaf, i.e., three stem-loop structures enclosed by a long distance interaction (LDI). This LDI prevents translation because its 3' moiety contains the Shine-Dalgarno sequence of the maturation gene. Previously, several observations suggested that folding of the cloverleaf is kinetically delayed, providing a time window for ribosomes to access the RNA. Here we present direct evidence for this model. In vitro experiments show that ribosome binding to the maturation gene is faster than refolding of the denatured cloverleaf. This folding delay appears related to special properties of the leader sequence. We have replaced the three stem-loop structures by a single five nt loop. This change does not affect the equilibrium structure of the LDI. Nevertheless, in this construct, the folding delay has virtually disappeared, suggesting that now the RNA folds faster than ribosomes can bind. Perturbation of the cloverleaf by an insertion makes the maturation start permanently accessible. A pseudorevertant that evolved from an infectious clone carrying the insertion had overcome this defect. It showed a wild-type folding delay before closing down the maturation gene. This experiment reveals the biological significance of retarded cloverleaf formation.

Base Sequence↗
PubMed1996

Rho-dependent termination of transcription is governed primarily by the upstream Rho utilization (rut) sequences of a terminator.

A Rho-dependent transcription terminator in Escherichia coli DNA consists of an upstream part for Rho utilization (rut) and the transcription stop point (tsp) region. To test the role of the tsp region variants of the coliphage lambda cro gene terminator, tR1, containing inserts of non-terminator sequences between its rut and tsp regions were tested for termination function. The results showed that termination occurred with high efficiency at multiple sites in each of the new sequences with the positions of the sites coinciding with transcriptional pause points in the insert sequence and that the efficiency of termination was not directly proportional to the extent of pausing at those points. Thus, in contrast to the rut sequences, which are relatively rare in DNA, many different sequence segments can function as a tsp region. Studies with isolated transcripts showed that a rut element and sequences 3' of the rut element were both needed to activate ATP hydrolysis by Rho factor with the degree of activation depending on the length and the specific sequence of the 3' segment. These results support models for Rho action in which ATP hydrolysis is coupled to interactions of Rho protein with RNA 3' of the rut region.

Adenosine Triphosphatases↗
PubMed1996

Protein-protein interactions involving T4 phage-coded deoxycytidylate deaminase and thymidylate synthase.

The enzymes deoxycytidylate deaminase (EC) and thymidylate synthase (EC) are functionally associated with one another, since they catalyze sequential reactions. In T4 coliphage infection the two enzymes are found in dNTP synthetase, a multienzyme complex for deoxyribonucleotide biosynthesis. Protein-protein interactions involving the phage-coded forms of these two enzymes have been explored in three experiments that use the respective purified protein as an affinity ligand. First, an extract of radiolabeled T4 proteins was passed through a column of immobilized enzyme (either dTMP synthase or dCMP deaminase), and the specifically bound proteins were identified. Second, two mutant form of dCMP deaminase (H90N and H94N), altered in presumed zinc-binding sites, were analyzed similarly, with the results suggesting that some, but not all, interactions require normal structure near the catalytic site. Third, affinity chromatography using either enzyme as the immobilized ligand, revealed interactions between the two purified enzymes in the absence of other proteins. In these experiments we noted a significant effect of dCTP, an allosteric modifier of dCMP deaminase, upon the interactions.

Bacteriophage T4↗
PubMed1996

Interactions between a minimal protein serine/threonine phosphatase and its phosphopeptide substrate sequence.

The protein phosphatase encoded by coliphage lambda (PPlambda) was found to be the equivalent of the minimal catalytic core of serine/threonine protein phosphatases (PP) by biochemical and mutational criteria. Bacterially expressed truncated versions of PP1 and PP5 phosphatases, representing the catalytic cores homologous to PPlambda, exhibited potent phosphatase activity. Unlike full-length PP1, but like PPlambda, the recombinant cores could use casein, p-nitrophenyl phosphate, and a wide variety of peptides as substrates and were resistant to okadaic acid, microcystin-LR, and trypsin. Mutations of His173, Asp208, or Arg221 had little effect on the activity of the PP1 core protein, indicating its closer identity with PPlambda than with full-length PP1. Terminal deletions of a few amino acids of the cores destroyed their activity, supporting their minimal nature. Analysis of PPlambda mutants suggested an influence of the substrate on metal ion binding. The minimal length of a phosphopeptide substrate of PPlambda appeared to be a phosphorylated serine/threonine flanked by 1 or 2 amino acid residues on either side, the N-terminal ones being more effective.

Amino Acid Sequence↗