Biochemical and genetic analysis of operator contacts made by residues within the beta-sheet DNA binding motif of Mnt repressor

EMBO J. 1992 Jan;11(1):215-23.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • Binding Sites
  • Coliphages / chemistry
  • Coliphages / genetics*
  • DNA Mutational Analysis
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / genetics*
  • Escherichia coli / genetics
  • Methylation
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • Operator Regions, Genetic / genetics*
  • Repressor Proteins / chemistry
  • Repressor Proteins / genetics*
  • Sequence Homology, Nucleic Acid
  • Viral Proteins / chemistry
  • Viral Proteins / genetics*
  • Viral Regulatory and Accessory Proteins

Substances

  • DNA-Binding Proteins
  • Repressor Proteins
  • Viral Proteins
  • Viral Regulatory and Accessory Proteins
  • phage repressor proteins