Biochemical and genetic analysis of operator contacts made by residues within the beta-sheet DNA binding motif of Mnt repressor.
Residues 2, 6, 8 and 10 of Mnt repressor are the major determinants of operator DNA binding and recognition. Here, we investigate the interaction of wild-type Mnt and mutants bearing the Arg2----Lys, His6----Ala, Asn8----Ala and Arg10----Lys mutations with operator DNA modified by methylation or by symmetric base substitutions. The wild-type pattern of methylation interference is altered in specific ways for each of the mutant proteins. In addition, some of the mutant proteins show a 'loss of contact' phenotype with specific mutant operators. Taken together, these and previous results predict the following contacts between side chains in the Mnt tetramer and operator DNA: Arg2 recognizes the guanines at operator positions 10 and 12; His6 contacts the guanines at operator positions 5 and 17; Asn8 contacts operator positions 4, 7, 15 and 18; Arg10 contacts the guanines at operator positions 8 and 14. The proposed contacts can be accommodated in a structural model in which the anti-parallel beta-sheet motifs of Mnt dimers lie in the major grooves of each operator half-site, centered over pseudo-symmetry axes that are 5.5 bp from the central dyad axis of the operator.
Knight
K L
KL
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Sauer
R T
RT
D001483:Base Sequence; D001665:Binding Sites; D003090:Coliphages / Q000737:chemistry / Q000235:genetics*; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D008745:Methylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009875:Operator Regions, Genetic / Q000235:genetics*; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics*; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins