Mutational analysis of the lac regulatory region: second-site changes that activate mutant promoters.
Second-site mutations that restored activity to severe lacP1 down-promoter mutants were isolated. This was accomplished by using a bacteriophage f1 vector containing a fusion of the mutant E. coli lac promoters with the structural gene for chloramphenicol acetyltransferase (CAT), so that a system was provided for selecting phage revertants (or pseudorevertants) that conferred resistance of phage-infected cells to chloramphenicol. Among the second-site changes that relieved defects in mutant lac promoters, the only one that restored lacP1 activity was a T----G substitution at position -14, a weakly conserved site in E. coli promoters. Three other sequence changes, G----A at -2, A----T at +1, and C----A at +10, activated nascent promoters in the lac regulatory region. The nascent promoters conformed to the consensus rule, that activity is gained by sequence changes toward homology with consensus sequences at the -35 and -10 regions of the promoter. However, the relative activities of some promoters cannot be explained solely by consideration of their conserved sequence elements.
Rothmel
R K
RK
Department of Biochemistry, University of Rochester School of Medicine and Dentistry, NY 14642.
LeClerc
J E
JE
D001483:Base Sequence; D003090:Coliphages / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D007763:Lac Operon; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic
D012333:RNA, Messenger