Characterization of regulatory circuit controlled by the purine repressor protein in Escherichia coli
Abstract
The regulatory circuit controlled by the purine repressor in Escherichia coli was studied by gene fusion and cloning. The expression of genes in the circuit was determined in isogenic purR and $purR\sp+$ strains. These measurements indicated 5 to 17 fold coregulation of genes purF, purHD, purC, purMN, purL and pur EK and thus confirm the existence of a pur regulon. Each locus of the pur regulon contains a 16 base pair conserved operator sequence that overlaps with the promoter. The purR product, purine repressor, was shown to bind specifically to each operator. Thus, binding of repressor to each operator negatively coregulates expression by inhibiting transcription initiation. The purB gene of E. coli, which encodes adenylosuccinate lyase, was cloned by functional complementation of a purB mutant. A single purB operator is 224 base pairs downstream of the transcription start site and overlaps with codons 62 to 67 in the protein coding region. Transcriptional regulation of purB involves binding of purine repressor to the 16 bp conserved operator. This regulation is only 2 to 3 fold. A promoter replacement experiment and in vivo RNA analysis indicated that repressor-operator control is independent of both the purB promoter and other 5$\sp\prime$ flanking sequences. Thus, binding of repressor to the purB operator inhibits transcription elongation by a roadblock mechanism. By assay of adenylosuccinate lyase activity, purA was found to be regulated 2 to 3 fold by purR and about 2 fold by a purR independent mechanism. Two operator sites were identified in the 5$\sp\prime$ flanking region which bind purine repressor in vitro and are required for in vivo regulation. By computer analysis, the sequences similar to pur operators were identified in promoter region of glnB, prsA, and speA. The purine repressor interacts with each of the operators and represses expression of these genes 2 to 3 fold. These genes have functions related to the nucleotide metabolism. It is now established that all genes for synthesis of purine nucleotides in E. coli respond to pools of purine bases via PurR, with genes dedicated to de novo synthesis of IMP exhibiting a greater response than those involved in the branches to AMP and GMP.
Degree
Ph.D.
Advisors
Zalkin, Purdue University.
Subject Area
Biochemistry
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