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Nucleic Acids Research, 1989, Vol. 17, No. 10 3909-3925
© 1989

MOLECULAR BIOLOGY

Mutational analysis of the lac regulatory region: second-site changes that activate mutant promoters

Randi Kubrick Rothmel+ and J.Eugene LeClerc*

Department of Biochemistry, University of Rochester School of Medicine and Dentistry Rochester, NY 14642, USA

*To whom correspondence should be addressed

Received December 19, 1988. Revised April 21, 1989. Accepted April 21, 1989.

Second-site mutations that restored activity to severe lacPl down-promoter mutants were isolated. This was accomplished by using a bacteriophage fl vector containing a fusion of the mutant E. coil lac promoters with the structural gene for chlorampnenicol acetyltransferase (CAT), so that a system was provided for selecting phage revertants (or pseudorevertants) that conferred resistance of phage-infectedcells to chloramphenicol. Among the second-site changes that relieved defects in mutant lac promoters, the only one that restored lacPl 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 activty is gainedoy 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.

+Present address: Department of Microbiology and Immunology, University of Illinois at Chicago, College of Medicine, Chicago, IL 60680, USA


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