The bacteriophage D108 Ner repressor binds a conformationally distinct operator.
The Ner protein encoded by the transposable coliphage D108, an 8.6 kDa lambda Cro-like repressor, binds to an operator spanning 50 bp of DNA. The distinguishing features of this operator are two perfect 11-bp inverted repeats (5'-CCGTGAGCTAC-3') that are separated by an 8-bp AT-rich spacer. Hyperreactivity of the ner operator to potassium permanganate and the hydroxyl radical indicate that the AT-rich spacer assumes a variant conformation consistent with a bend. Using an electrophoretic mobility shift assay, we demonstrated that Ner does not display significant affinity for a single 11-bp site. Furthermore, DNase I protection analysis and circular-permutation binding assays reveal that alterations in the length and sequence of the AT-rich spacer that separates the 11-bp inverted repeats significantly alter Ner-operator interactions, and demonstrate that the intrinsically bent ner operator is conformationally altered upon protein binding.
Kukolj
G
G
Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
DuBow
M S
MS
D001435:Bacteriophages / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009876:Operon; D012091:Repetitive Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000378:metabolism*; D015183:Restriction Mapping; D019895:Transposases / Q000235:genetics; D014764:Viral Proteins / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins
D004275:DNA, Ribosomal; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D019895:Transposases