The roles of residues 5 and 9 of the recognition helix of Lac repressor in lac operator binding.
We constructed expression libraries for Lac repressor mutants with amino acid exchanges in positions 1, 2, 5 and 9 of the recognition helix. We then analysed the interactions of residues 5 and 9 with operator variants bearing single or multiple symmetric base-pair exchanges in positions 3, 4 and 5 of the ideal fully symmetric lac operator. We isolated 37 independent Lac repressor mutants with five different amino acids in position 5 of the recognition helix that exhibit a strong preference for particular residues in position 2 and, to a lesser extent, in position 1 of the recognition helix. Our results suggest that residue 5 of the recognition helix (serine 21) contributes to the specific recognition of base-pair 4 of the lac operator. They further suggest that residue 9 of the recognition helix (asparagine 25) interacts non-specifically with a phosphate of the DNA backbone, possibly between base-pairs 2 and 3.
Sartorius
J
J
Institut für Genetik, Universität zu Köln, Germany.
Lehming
N
N
Kisters-Woike
B
B
von Wilcken-Bergmann
B
B
Müller-Hill
B
B
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015723:Gene Library; D007763:Lac Operon / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D009875:Operator Regions, Genetic; D012097:Repressor Proteins / Q000378:metabolism*
D000596:Amino Acids; D004269:DNA, Bacterial; D012097:Repressor Proteins