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  • Title: Regulation of the F plasmid traY promoter in Escherichia coli K12 as a function of sequence context.
    Author: Silverman PM, Wickersham E, Rainwater S, Harris R.
    Journal: J Mol Biol; 1991 Jul 20; 220(2):271-9. PubMed ID: 1906941.
    Abstract:
    TraJ and SfrA are, respectively, plasmid and host (Escherichia coli)-encoded proteins normally required for F plasmid traY promoter function. Beginning with plasmids in which a traY-lacZ fusion gene, designated phi (traY'-'lacZ)hyb, and lacY are expressed from the F plasmid traY promoter, we isolated mutants in which lac gene expression was SfrA or TraJ-independent. A total of 45 of 50 SfrA-independent isolates obtained after 2-aminopurine mutagenesis proved to have chromosomal mutations, whereas four out of four isolates obtained without mutagenesis had plasmid mutations. All of 17 isolates selected for TraJ-independent expression after mutagenesis had plasmid mutations. By restriction endonuclease digestions, 25 of 26 SfrA-independent and TraJ-independent plasmid mutations were insertions. Four of the former and three of the latter were examined further. By sequence analysis, all seven proved to be IS1 or IS2 insertions defining five insertion sites between base-pairs -49 and -82 with respect to the major traY transcription initiation site. In two cases, the same insertion allele was obtained from the two selection schemes. All three of the mutants selected for TraJ-independent gene expression manifested SfrA-independent expression as well, and levels of beta-galactosidase in different plasmid mutant strains lacking TraJ and SfrA were indistinguishable. By primer extension analysis, transcription initiation sites for traY mRNA synthesis were unaltered by the mutations. Replacing the tra sequence upstream from base-pair -78, without genetic selection, increased beta-galactosidase activity in the absence of TraJ and SfrA greater than tenfold. Activity increased two- to threefold more in a traJ+ sfrA mutant strain, and fivefold more in a traJ+ sfrA+ strain. Activity was unaltered in an sfrA+ strain without TraJ. By primer extension analysis, the traY promoter was utilized under all conditions. The data indicate that regulation of traY promoter activity is strongly dependent on sequence context.
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