THE MOLECULAR BASIS OF PROMOTER ACTIVITY AT A SITE WITHIN THE TRYPTOPHAN OPERON OF ESCHERICHIA COLI
Abstract
Within the structural genes of the tryptophan operon of Escherichia coli there exist several sites where high efficiency constitutive promoters can arise through mutation (McPartland and Somerville, J. Bacteriol. 128 557 (1976)). When transcription is initiated at these sites there is a 15 to 20 fold increase in the level of downstream enzymes. One such promoter, trpD11, lies within the trpD gene and was first recognized through an ochre (UAA) mutation. A family of different codons derived from UAA exist at this site, with approximately 60% retaining promoter activity (Hausler, Ph.D. Thesis, 1978 Purdue University). Five promoter-active and two promoter-inactive trpD11 alleles were cloned from appropriately marked pPF102 plasmids (M..w 1.12 x 10('8)) into the unique HindIII site of pBR322.12. The position of the trpD11 site within the 2.8 kb HindIII fragment was determined via deletion mapping of the plasmid containing the trpD11 (UAA) allele. DNA sequencing confirmed previous genetic results that the promoter phenotype of trpD11 (UAA) arose as a result of a CG (--->) TA transition. The remainder of the region is identical to the wild type sequence. A good correlation exists between the trpD11 region and the Rosenberg and Court prototype promoter sequence (Rosenberg and Court, Ann. Rev. Genet. 13 319 (1979)). The sequence of the promoter-inactive trpD11 alleles also conforms to the prediction that most promoter down mutations of the -10 region (23 of the 31 sequenced to date) occur in one of the three most conserved positions of this region. Several putative CAP binding sites were also noted in the trpD11 region. Double mutant strains were then constructed which contain mutations in the crp locus to examine the possible role of the CRP protein on the activity of the trpD11 promoters. In vivo, the presence of a nonfunctional catabolite activator protein enhances the level of transcription from the trpD11 promoter.
Degree
Ph.D.
Subject Area
Biology
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