title: Purification and DNA-binding properties of the cro-type regulatory repressor protein cng encoded by the Lactobacillus plantarum phage phi g1e.
abstract: The putative repressor protein Cng (10kDa on an SDS gel) for the lytic pathway of Lactobacillus plantarum phage &phi;g1e was purified using the Escherichia coli Pt7 system, and its DNA-binding ability for the seven operator-like sequences, the GATAC-boxes (Gb1 to Gb7), was investigated in vitro. In gel-shift assays, Cng selectively bound to the DNA fragments containing the GATAC-box(es). In addition, DNase I footprinting analysis with supercoiled DNA demonstrated that Cng can specifically cover about a 25bp region centered around each of the GATAC-boxes, although two boxes, Gb4 and Gb6, were only partially protected. Moreover, protein crosslinking experiments using glutaraldehyde suggested that Cng most likely functions as a dimer. On the other hand, the binding ability of Cpg for the GATAC-boxes in supercoiled DNA was also examined under the same conditions as in Cng; unlike Cng, Cpg covered Gb4 and Gb6 completely sufficiently as well as the other five boxes. Thus, the present and previous [Kakikawa et al., Gene 215 (1998) 371-379; 242 (2000) 155-166] results indicate a possibility that the two proteins Cng and Cpg selectively bind to the GATAC-boxes that act as operators, and can decide between the lytic or lysogenic pathways through repression of the promoter activity of P(R) as well as P(L).
authors.affiliation: Molecular Biology Group, Toyama University, Gofuku, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Kakikawa
authors.forename: S
authors.initials: S
authors.lastname: Ohkubo
authors.forename: T
authors.initials: T
authors.lastname: Sakate
authors.forename: M
authors.initials: M
authors.lastname: Sayama
authors.forename: A
authors.initials: A
authors.lastname: Taketo
authors.forename: K
authors.initials: K
authors.lastname: Kodaira
mesh_terms: D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003432:Cross-Linking Reagents; D018983:DNA Footprinting; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D003850:Deoxyribonuclease I; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D007778:Lactobacillus / Q000821:virology*; D008969:Molecular Sequence Data; D012097:Repressor Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: C114486:Cng protein, Bacteriophage phig1e; D003432:Cross-Linking Reagents; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D017361:Viral Nonstructural Proteins; D003850:Deoxyribonuclease I