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 Title: Functional organization, nucleotide sequence and regulation of the Bacillus subtilis pyrimidine biosynthetic operon Author(s): Quinn, Cheryl Lee Doctoral Committee Chair(s): Switzer, Robert L. Department / Program: Biochemistry Discipline: Biochemistry Degree Granting Institution: University of Illinois at Urbana-Champaign Degree: Ph.D. Genre: Dissertation Subject(s): Biology, Molecular Biology, Microbiology Chemistry, Biochemistry Abstract: A 12.5 kilobase segment of Bacillus subtilis chromosomal DNA containing the entire pyrimidine biosynthetic (pyr) gene cluster has been cloned and sequenced. The cloning of a 10.5 kb PstI fragment of the B. subtilis chromosome containing the pyr genes was previously reported (Lerner et al., 1987, J. Bacteriol. 169, 2202-2206). The sequence of the entire PstI fragment is reported here. In this study, a chromosomal fragment that overlapped the 5$\sp\prime$ end of the PstI fragment and extended to an EcoRI site that was 5 kb upstream was cloned by plasmid rescue. The nucleotide sequence for 2 kb of the newly cloned DNA was also determined. The sequenced DNA has seven cistrons encoding the six enzymes of de novo pyrimidine nucleotide biosynthesis and two open reading frames of unknown function. Based on the sequence and mapping of transcripts, the genes in this cluster appear to be transcribed on one large polycistronic message in the order ORF1, pyrB, pyrC, pyrAA, pyrAB, ORF2, pyrD, pyrF, pyrE. The deduced amino acid sequences for six pyrimidine biosynthetic enzymes from B. subtilis and comparisons to the corresponding sequences from numerous other species are presented. The 3$\sp\prime$ ends of the reading frames overlap the 5$\sp\prime$ ends of the downstream open reading frames for all cistrons in the cluster except ORF1 and pyrB, which are separated by a 145 base pair intercistronic region. The start of transcription was mapped by primer extension to a G residue 158 nucleotides upstream from the translation initiation codon of ORF1. This site is preceded by a typical B. subtilis sigma A dependent promoter. A promoter indicator plasmid was used to show that this region carried a promoter. Transcription from this promoter was regulated by pyrimidines. Analysis of the nucleotide sequence between the start of transcription and the start of the ORF1 gene reveals a region of dyad symmetry followed by a series of T residues. This putative rho-independent terminator was shown to be important for pyrimidine regulation of the marker gene on the promoter indicator plasmids. No transcripts initiating from the intercistronic space between ORF1 and pyrB were detected with S1 nuclease mapping; however, a transcription terminator was detected in this region that reduced but did not fully block transcriptional readthrough. This terminator was not regulated by pyrimidines in the growth medium under the conditions tested. The role, if any, of this transcription terminator in the regulation of pyr operon expression is yet to be determined. The presence of a recognition sequence for the Spo0A protein 100 nucleotides upstream from the transcription intitiation site of the pyr operon suggests a possible role for this protein in the developmental regulation of the pyr operon. Issue Date: 1991 Type: Text Language: English URI: http://hdl.handle.net/2142/19626 Rights Information: Copyright 1991 Quinn, Cheryl Lee Date Available in IDEALS: 2011-05-07 Identifier in Online Catalog: AAI9136704 OCLC Identifier: (UMI)AAI9136704
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