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 wa...
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Veröffentlicht in: | Nucleic acids research 1989-05, Vol.17 (10), p.3909-3925 |
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Sprache: | eng |
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Zusammenfassung: | 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. |
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ISSN: | 0305-1048 1362-4962 |
DOI: | 10.1093/nar/17.10.3909 |