Structure, Regulation and Comparative Biochemistry of the Pyrimidine Biosynthetic Gene Cluster in Bacillus Subtilis (Atcase)

219 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1987.A 10.5 kilobase PstI endonuclease fragment encoding the entire Bacillus subtilis pyrimidine biosynthetic (pyr) gene cluster was cloned in Escherichia coli. The cloned fragment also complemented E. coli pyrB, C, D, E, and F mutants. A restriction map of the cluster was generated, and many subclones were analyzed for their ability to complement E. coli pyr mutants. From these data a genetic map was generated; the gene order is pyrBCADFE. The B. subtilis pyrB gene was shown to be expressed in E. coli on the basis of immunochemical properties of the aspartate transcarbamylase (ATCase) activity in extracts.ATCase from B. subtilis is a catalytic trimer that contains no regulatory subunits and is not subject to allosteric regulation. Thus, comparison of its structure to that of E. coli ATCase can illuminate elements potentially involved in catalysis and regulation of ATCases. The B. subtilis ATCase gene (pyrB) was subcloned from the 10.5 kilobase PstI fragment by complementation of an E. coli pyrB host and sequenced by the method of Maxam and Gilbert. The subunit size (34,185 daltons), N- and C-terminal sequence and amino acid composition predicted by the DNA sequence agreed very well with the values obtained with the purified enzyme. Sequences of B. subtilis and E. coli ATCases were 44% conserved in the polar (N-terminal) domain and 27% conserved in the equatorial domain. Of 14 amino acid residues identified in E. coli ATCase by X-ray crystallography as in the active site, 12 (Ser-52, Arg-54, Thr-55, Ser-80, Lys-84, Arg-105, Gly-128, His-134, Gln-137, Arg-167, Arg-229, Gln-231) are conserved in B. subtilis ATCase. Two such residues (Thr-168 and Leu-267) are not conserved. The tertiary folding of the two ATCases appears to be similar, but the subunit:subunit interactions are not obviously conserved.The pyrB gene is flanked on both the 5' and 3' sides by open reading frames that overlap the pyrB coding region by 8 and 5 codons, respectively. The sequence of the 5' flanking region to B. subtilis pyrB suggests that repression of the enzyme may involve attenuation as with E. coli pyrBI. Pyrimidine repression of B. subtilis pyrB gene expression occurs at the transcriptional level.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD