Autogenous transcriptional attenuation of de novo pyrimidine biosynthesis in Bacillus subtilis
Turner, Robert Joseph
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Permalink
https://hdl.handle.net/2142/20057
Description
Title
Autogenous transcriptional attenuation of de novo pyrimidine biosynthesis in Bacillus subtilis
Author(s)
Turner, Robert Joseph
Issue Date
1996
Doctoral Committee Chair(s)
Switzer, Robert L.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Biology, Microbiology
Chemistry, Biochemistry
Language
eng
Abstract
The genes encoding the enzymes for de novo pyrimidine biosynthesis in the gram-positive microorganism Bacillus subtilis were previously shown to be clustered at 139 min on its chromosome, transcribed from a single promoter and regulated at the level of transcription by the availability of pyrimidines.
A complete transcript of the B. subtilis pyr operon contains the following elements in 5$\sp\prime$ to 3$\sp\prime$ order: a 151 nt untranslated leader; pyrR, encoding a 20 kDa protein; a 173 nt intercistronic region; pyrP, encoding a 46 kDa protein; a 145 nt intercistronic region; followed by 8 overlapping cistrons encoding all of the 6 enzymes for de novo pyrimidine biosynthesis. Transcription is controlled by the availability of pyrimidines via an attenuation mechanism. Computer analysis of potential RNA structures revealed that there are three rho-independent transcription terminators within the operon, each of which is preceded by another stem-loop structure, the antiterminator, whose formation would prevent formation of the terminator stem-loop. These are located in the leader, pyrR-pyrP intercistronic, and pyrP-pyrB intercistronic regions. Each antiterminator contains a 50 base conserved sequence in its promoter proximal half. Various transcriptional fusions of the pyr promoter and surrounding sequences to promoterless reporter genes support an attenuation mechanism whereby when pyrimidines are abundant, the PyrR protein binds to the conserved sequence in the pyr mRNA and disrupts the antiterminator, permitting terminator hairpin formation and promoting transcription termination.
The regulatory activity of PyrR was confirmed when deletion of pyrR from the chromosome resulted in the constitutive elevated expression of aspartate transcarbamylase, which is encoded by pyrB, the third gene in the operon. Complementation of an E. coli upp mutant, as well as direct enzymatic assay, has demonstrated that pyrR also confers uracil phosphoribosyltransferase (UPRTase) activity. Analysis of pyrR and upp deletion mutants demonstrated that upp, not pyrR, encodes the quantitatively important UPRTase activity. Complementation of B. subtilis furA2, a mutant defective in uracil transport, and amino acid sequence alignment with the E. coli uracil permease strongly suggests pyrP encodes an integral membrane uracil permease.
This work has made it possible to biochemically characterize the regulation of the B. subtilis pyr operon. The availability of purified PyrR permits the analysis of the RNA-binding and UPRTase properties of PyrR, the reconstruction of B. subtilis pyr attenuation in vitro, and the determination of the high resolution structure of PyrR. (Abstract shortened by UMI.)
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