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PubMed1996

Purification and self-association equilibria of the lysis-lysogeny switch proteins of coliphage 186.

The CI repressor protein, responsible for maintenance of the lysogenic state, and the Apl protein, required for efficient prophage induction, are the two control proteins of the lysis-lysogeny transcriptional switch of coliphage 186. These proteins have been overexpressed, purified, and their self-association behavior examined by sedimentation equilibrium. Phage 186 CI dimers self-associate in solution through tetramers to octamers in a concerted process. The Apl protein of 186 is an unusual example of a helix-turn-helix protein which is monomeric in solution.

Base Sequence↗
PubMed1996

DNA binding by the coliphage 186 repressor protein CI.

The cI gene of coliphage 186 maintains lysogeny and confers immunity to 186 infection by repressing the major early promoter, p(R), and the promoter for the late transcription activator gene, p(B). Gel mobility shirt and DNase I footprinting show that CI protein binds to the DNA at p(R) and p(B) and also to sites approximately 300 base pairs upstream and downstream of p(R), called FL and FR. Mutations which cause virulence reduce CI binding to p(R). The biochemical and genetic data identify three CI operators at p(R), two at p(B), and single operators at FL and FR. The operators at the p(B), FL, FR, and central p(R) sites are inverted repeat sequences, separated by 5 base pairs (Type A) or, in the case of p(R), by 4 base pairs (Type A'). A different inverted repeat operator sequence (Type B) is proposed for the binding sites on each side of the central site at p(R). Thus, CI appears to recognize two distinct DNA sequences. CI binds cooperatively to adjacent operators, and binding at p(R) is strongly dependent on these cooperative interactions. A high order CI multimer appears to be the active DNA binding species, even at single operators.

Amino Acid Sequence↗
PubMed2001

The structure of the coliphage HK022 Nun protein-lambda-phage boxB RNA complex. Implications for the mechanism of transcription termination.

Nun protein from coliphage HK022 binds to phage boxB RNA and functions, in contrast to phage lambda N protein, as a transcriptional terminator. The basic Nun-(10-44) peptide contains the boxB RNA binding arginine rich motif, ARM. The peptide binds boxB RNA and competes with the phage lambda ARM peptide N-(1-36) as indicated by nuclear magnetic resonance (NMR) spectroscopy titrations. In two-dimensional nuclear Overhauser enhancement spectroscopy experiments boxB RNA in complex with Nun-(20-44) exhibits the same pattern of resonances as it does in complex with N peptides containing the ARM, and we could show that Nun-(20-44) forms a bent alpha-helix upon binding to the boxB RNA. The structure of the boxB RNA-bound Nun-(20-44) was determined on the basis of 191 intra- and 30 intermolecular distance restraints. Ser-24 is anchored to the lower RNA stem, and stacking of Tyr-39 and A7 is clearly experimentally indicated. Arg-28 shows numerous contacts to the RNA stem. Leu-22, Ile-30, Trp-33, Ile-37, and Leu-41 form a hydrophobic surface, which could be a recognition site for additional host factors such as NusG. Such a hydrophobic surface area is not present in N-(1-36) bound to boxB RNA.

Amino Acid Sequence↗
PubMed2001

The helix-turn-helix motif of the coliphage 186 immunity repressor binds to two distinct recognition sequences.

The CI protein of coliphage 186 is responsible for maintaining the stable lysogenic state. To do this CI must recognize two distinct DNA sequences, termed A type sites and B type sites. Here we investigate whether CI contains two separate DNA binding motifs or whether CI has one motif that recognizes two different operator sequences. Sequence alignment with 186-like repressors predicts an N-terminal helix-turn-helix (HTH) motif, albeit with poor homology to a large master set of such motifs. The domain structure of CI was investigated by linker insertion mutagenesis and limited proteolysis. CI consists of an N-terminal domain, which weakly dimerizes and binds both A and B type sequences, and a C-terminal domain, which associates to octamers but is unable to bind DNA. A fusion protein consisting of the 186 N-terminal domain and the phage lambda oligomerization domain binds A and B type sequences more efficiently than the isolated 186 CI N-terminal domain, hence the 186 C-terminal domain likely mediates oligomerization and cooperativity. Site-directed mutation of the putative 186 HTH motif eliminates binding to both A and B type sites, supporting the idea that binding to the two distinct DNA sequences is mediated by a variant HTH motif.

Amino Acid Sequence↗
PubMed1975

Modification of E. coli ribosomes and coliphage MS2 RNA by bisulfite: effects on ribosomal binding and protein synthesis.

The reaction of E. coli 70s ribosomes with 0.2 M NaH-35 s03 (pH 7.1, 3.5hrs, 37 degree) led to the conversion of 4.5% of the uracil residues of the R, RNA into 5.6-dihydrouracil-6-sulfonate residues. The modified ribosomes exhibited a significant decrease in their ability to bind (14-C)-phenylalanyl-(RNA-phe and to incorporate (14-C)-phenylalanine into protein in the presence of polyuridylic acid. The ability of the modified ribosomes to form an initiation complex as measured by the A-U-G or coliphage MS2 RNA dependent binding of (14-C)-fmet-tRNA-fmet was also impaired, as was their ability to incorporate (14-C) lysine into protein with MS2 RNA as messenger. Treatment os MS RNA with 0.2 M sodium (35-S) bisulfite, pH 7.0 at 25 degrees C resulted in the substitution of 2.7% and 6.2% of the uracil residues by bisulfite after 1 and 3.5 hrs of reaction, respectively. Impairment of function of the MS2 RNA in both initiation complex formation and transplantation assays was observed. These reactions of uracil residues of mRNA and rRNA may be a cause of biological damage inflicted by sodium bisulfite and sulfur dioxide.

Bacterial Proteins↗
PubMed1999

Use of a linear multicopy vector based on the mini-replicon of temperate coliphage N15 for cloning DNA with abnormal secondary structures.

A new cloning vector pN15L is described. It is a linear 13.8 kb plasmid based on the coliphage N15 mini-replicon. The vector capacity exceeds 50 kb and the copy number is 250 per Escherichia coli chromosome. We show that some artificial and natural palindromes and approximately 5% of human DNA Bgl II fragments can be cloned effectively in linear vector pN15L, whereas they either sharply reduce the copy number of circular vector pUC19 or cannot be cloned at all. We conclude that pN15L may be usefully employed to clone large imperfect palindromes and some abnormal sequences of human DNA.

Animals↗
PubMed1989

Artificial immune system against viral infection involving antisense RNA targeted to the 5'-terminal noncoding region of coliphage SP RNA.

We previously reported the utilization of antisense RNA in the development of a novel immune system against RNA coliphage SP proliferation (Hirashima et al. [1986] Proc. Natl. Acad. Sci. U.S. 83, 7726-7730). We attempted to determine the most effective (i.e., those eliciting antiviral activity) sequences for targeting micRNAs within the 5'-terminal noncoding region of 54 nucleotides (nt). It was found that a 30-nt micRNA against the sequence from base 32 to 61 exhibited nearly complete inhibition of phage production. Upon further dissection of this sequence, it was concluded that the most effective micRNA against phage SP production should contain the sequences complementary to the Shine-Dalgarno (SD) sequence of the first gene and its 13-nt upstream sequence. The addition of downstream sequences had little effect. These results suggest that the micRNA functions by preventing the binding of ribosomes to the SD sequence of the first gene. The addition of further upstream sequences had a significant negative effect on the micRNA function, indicating that the removal of such impeditive sequences from a micRNA is an important strategy for the development of a potent micRNA immune system.

Base Sequence↗
PubMed1986

The complete nucleotide sequence of the group II RNA coliphage GA.

The complete nucleotide sequence of the RNA coliphage GA, a group II phage, is presented. The entire genome comprises 3466 bases. Three large open reading frames were identified, which correspond to the maturation protein gene (390 amino acids), the coat protein gene (129 amino acids) and the replicase beta-subunit protein gene (531 amino acids). In addition, untranslated regions occur at the 5' (135 bases) and 3' (122 bases) ends of the molecule. Two intercistronic untranslated regions occur between the cistrons for the maturation and coat proteins, and between the coat and beta-subunit proteins. We have compared the nucleotide sequence of GA RNA with the published sequence of MS2 RNA, and show that they are related. The comparative structures of two important regulatory regions are presented; the coat protein binding site which is involved in translational repression of the replicase beta-subunit protein gene, and a hairpin in a region proximal to the lysis protein gene.

Base Sequence↗
PubMed1987

Structural relatedness of lysis proteins from colicinogenic plasmids and icosahedral coliphages.

The host-lysis-inducing functions of phi X174 protein E and MS2 protein L were recently shown to reside on the N-terminal and C-terminal halves of the two respective lysis proteins. In the present study it is shown that the small lysis proteins encoded in various colicinogenic plasmids share local sequence similarities and certain structural characteristics with the essential peptides of their coliphage-coded counterparts. Despite their dissimilar sizes and origins, it is suggested that the colicinogenic lysis proteins are functionally analogous and evolutionarily related to those of icosahedral single-stranded DNA and RNA phages.

Amino Acid Sequence↗
PubMed1975

Occurrence of polyamines in coliphages T5, phiX174 and in phage-infected bacteria.

The polyamine spermidine and the diamine putrescine have been detected in coliphages T5 and phiX174. Polyamines were identified by thin-layer chromatography and mass-spectrometry of dansyl derivatives, as well as by ion-exchange chromatography. In phiX174 phages, polyamines were sufficient to neutralize 0.5% of DNA phosphates. The polyamine content of T5 phages depended on growth media and purification procedures, but at least 1% of DNA phosphates were neutralized by polyamines. After infection, an increase in cellular polyamine was noticed. This increase paralleled variations in ornithine decarboxylase activity.

Carboxy-Lyases↗
PubMed1983

Biochemical characterization of RNA coliphage MX1.

In order to clarify the taxonomic status of an RNA coliphage, MX1 (a serological intermediate between groups III and IV), we examined (i) read-through protein synthesis in a cell-free protein-synthesizing system, (ii) the peptide map of the coat protein and (iii) the RNA sequence in the 3'-terminal region of MX1 RNA. We found that the characteristics of MX1 were closer to those of group III phages than to those of group IV. For example, the gel elecrophoretic pattern of the protein coded for by MX1 RNA was the same as that of group III phage proteins. Peptide fingerprints of the coat protein of MX1 showed that seven tryptic peptides overlapped with corresponding peptides of Q beta (group III phage), whereas only two peptides overlapped with those of SP (group IV phage). Furthermore, in the base sequence of the first 200 nucleotides from the 3'-end of MX1 RNA, about 70% of the nucleotides were homologous to those of the Q beta RNA, whereas the homology to SP RNA was only 53%. These results suggest that MX1 is more closely related to group III phages than to group IV phages and we propose that it should be assigned to the former group.

Base Sequence↗
PubMed1985

On the evolution of the terminal redundancies of Klebsiella phage No. 11 and of coliphages T3 and T7.

The phylogenetic relationship between Klebsiella phage No. 11 and the classical coliphages T7 and T3, postulated in a previous study, was investigated at the nucleotide level by sequencing the termini of phage No. 11 DNA. This DNA was found to have a terminal redundancy of 181 base pairs. Comparison of the terminal sequences of T7, T3 and No. 11 DNA suggests that the terminal redundancies of the three phages contain different expansions and variations of the short 'founder sequence' 5' TTAACCTTGGG 3' of a common ancestor.

Bacteriophages↗
PubMed1977

Host factor for coliphage Qbeta RNA replication as an aid in elucidating phylogenetic relationships: the genus Pseudomonas.

Host Factor (HF) is a heat-stable RNA-binding protein required along with Qbeta replicase for in vitro transcription of coliphage Qbeta RNA. We have found that HF activity and antigenicity are conserved among certain Gram-negative bacterial species. We examined selected species within the genus Pseudomonas for the presence of the HF polypeptide's antigenicity and Qbeta RNA replication function. While we were unable to detect either of these in Pseudomonas diminuta or Pseudomonas vesicularis, the other eleven species tested contained cross-reacting material to Escherichia coli HF. Furthermore, in six of these eleven species we were able to detect HF activity. The detection of HF structure and function allowed the examined species to be grouped into three categories which we have called 'sets'. The results correlate well with those of previous studies on ribosomal RNA homology (Palleroni et al., 1973).

Antigens, Bacterial↗
PubMed1979

Continuous survey of the distribution of RNA coliphages in Japan.

In order to demonstrate the stability and continuity of RNA coliphages (phages) in their natural habitats, we investigated the amount and group types of RNA phages in sewage samples collected continuously from domestic drainage in Japan proper and islands in the seas adjacent to Japan (abbreviated simply as islands, hereafter) over a 5-yr period from 1973 to 1977. It was found that the frequencies of isolation of RNA phages were fairly high and constant. The group types of RNA phages isolated were also stable in the three cities. Choshi, Niigata, and Toyama in Japan proper. The average for the three cities was group II:III = 3:1. The investigation in islands revealed that the frequencies of isolation of RNA phages were fairly high as in the case of the above three cities in Japan proper and the group types of RNA phages isolated were also stable. That is to say, group II phages were predominant on Rishiri Island, Rebun I., Iki I., and Tsushima I., which are located relatively near to mainland Japan, while group III phages were predominant on Amamiohshima I., mainland Okinawa, Ishigakijima I., and Iriomotejima I., which are located south of Kyushu. It can thus be said that the RNA phages in the domestic drainage of Japan proper and islands remained more or less stable over at least the 5-yr period, and an apparent difference in the geographical distribution of RNA phages in Japan exists between Kyushu and Amamiohshima I.

Coliphages↗
PubMed1992

Control of gene expression in the temperate coliphage 186. IX. B is the sole phage function needed to activate transcription of the phage late genes.

Using plasmid clones we have determined that the late control function B is the only phage function that is needed to activate a late promoter of coliphage 186, and we predict that it functions as an auxiliary factor to RNA polymerase in the activation of late transcription. We have also shown that a high concentration of B will activate late transcription from a prophage, and we conclude that replicating DNA is not a template requirement for B to function. The original demonstration of a need for the replication gene A in late transcription can be explained by the fact that replication leads to an increase in B gene dosage, with the consequent increase in B concentration leading to the efficient activation of the late promoters.

Amino Acid Sequence↗
PubMed1993

The Cro-like Apl repressor of coliphage 186 is required for prophage excision and binds near the phage attachment site.

The Apl protein of the temperature coliphage 186 represses transcription of the immunity repressor gene and down-regulates lytic transcription. It is shown here that an apl- mutant is competent for lytic development and establishes lysogeny normally but is defective in excision of the prophage. The Apl protein binds between the lytic and lysogenic promoters and also near the phage attachment site, suggesting that its role in excision is direct. Apl thus appears to act as an excisionase as well as a repressor. The pattern of Apl-induced DNase I enhancements indicates that the DNA is bent by Apl. Potential Apl recognition sequences are identified; these sequences are directly repeated several times across each binding region and are spaced 10 or 11 bases apart, suggesting that Apl binds to one face of the DNA helix.

Attachment Sites, Microbiological↗
PubMed2000

Structure and function of a novel coliphage-associated sialidase.

A coliphage named 63D, isolated previously, associated sialidase as a component of phage particles. In order to localize the enzyme in phage particles, phages were partially destroyed by sonication, and the disrupted particles were size fractionated using a sucrose density gradient. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme assay and electron micrography of the fractions revealed the enzyme to be composed of four identical subunits with a molecular mass of 90 kDa, and the subunits were cross-linked by disulfide bonds. Electron micrographic observation indicated that six enzyme molecules were localized in a phage tail plate as a hexagonal array.

Cell Fractionation↗