Characterization of the Bacillus subtilis pur operon
Abstract
The Bacillus subtilis pur operon was characterized to better understand genetic organization and regulation in this gram positive bacterium. Over 13 kilobase pairs of DNA containing the pur operon were cloned and sequenced. Analysis of this sequence revealed 12 genes, eleven of which encode the nine enzymes required for de novo synthesis of IMP. The operon is organized as three overlapping sets of genes separated by intercistronic gaps followed by the last gene of the operon; purEKB-purC(orf)QLF-purMNH(J)-purD. Transcription of the operon is regulated by guanine and adenine. Guanine regulates transcription by a termination-antitermination mechanism in a 242 nucleotide mRNA leader. Regulation depends upon formation of RNA secondary structure in vivo. The structure of the RNA leader produced by guanine-regulated termination was deduced from analysis of intermediates in the decay of the terminated transcript. Adenine regulates transcription initiation by repression. Deletion analysis defined an element upstream of the promoter required for repression. A candidate repressor protein was partially purified from B. subtilis and was shown to bind to the control region of the pur operon. Expression and regulation throughout the operon was examined by using translational fusions of Escherichia coli lacZ to the first gene of each of the three clusters of overlapping genes. Measurements of $\beta$-galactosidase and mRNA provided preliminary data to suggest that a single promoter drives transcription of this operon. Regulation of transcription occurs only at the 5$\sp\prime$-end of the operon. Differential regulation of purC-lacZ enzyme suggested that a translational control may be present within the operon. Although levels of mRNA were similar at different sites throughout the operon, corresponding enzyme levels differed up to 7-fold. This difference in gene expression indicates that translational efficiencies may be important factors in determining the stoichiometry of the enzymes for de novo purine nucleotide synthesis.
Degree
Ph.D.
Advisors
Zalkin, Purdue University.
Subject Area
Microbiology
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