Open AccessAltered Sequences Changing the Operator-Binding Properties of the Lac Repressor: Colinearity of the Repressor Protein with the i -Gene Map
Author(s) -
Klaus Weber,
Terry Platt,
Don Ganem,
Jeffrey H Miller
Publication year - 1972
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.69.12.3624
Subject(s) - repressor , lac repressor , biology , alanine , point mutation , serine , amino acid , glutamine , nonsense mutation , yy1 , valine , biochemistry , microbiology and biotechnology , gene , leucine , binding site , genetics , missense mutation , mutation , gene expression , promoter , phosphorylation
A technique is described for mapping point mutations in the first 59 amino-acid residues of thelac repressor fromEscherichia coli , using less than 0.1 μmol (4 mg) of the purified protein. This technique was used to localize five mutations affecting the ability of thei -gene product to repressin vivo . These alterations are located at four different sites in the amino-terminal region of the repressor molecule. Three of these are missense mutations and result in changes from serine to proline (residue 16), threonine to alanine (residue 19), and alanine to valine (residue 53). Each amino-acid substitution alone is sufficient to eliminate repressionin vivo , presumably by altering the operator binding activity. The remaining two independently-isolated mutations are identical, and result in a change from a glutamine codon at position 26 to an amber (UAG) codon. Since suppression of this nonsense mutation with amber suppressors that insert leucine, tyrosine, serine, or glutamine restores repressor activity to the molecule, glutamine26 cannot be crucial for the operator-binding function. A comparison of the position of each altered residue with the genetic map enabled us to estimate the physical distance between several deletion-group endpoints.