Purification and DNA-binding properties of the cro-type regulatory repressor protein cng encoded by the Lactobacillus plantarum phage phi g1e.
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).
Kakikawa
M
M
Molecular Biology Group, Toyama University, Gofuku, Japan.
Ohkubo
S
S
Sakate
T
T
Sayama
M
M
Taketo
A
A
Kodaira
K
K
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
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