REGULATION OF THE BACILLUS-SUBTILIS PYRIMIDINE BIOSYNTHETIC (PYR) GENE-CLUSTER BY AN AUTOGENOUS TRANSCRIPTIONAL ATTENUATION MECHANISM

A complete transcript of the Bacillus subtilis pyr operon contains the following elements in 5' to 3' order: a 191-nucleotide (nt) untransla ted leader; pyrR, encoding a 20-kDa protein; a 173-nt intercistronic r egion; pyrP, encoding a 46-kDa protein; a 145-nt intercistronic region ; and eight overlapping cistrons encoding all of the six enzymes for d e novo pyrimidine biosynthesis. Transcription is controlled by the ava ilability of pyrimidines via an attenuation mechanism. There are three transcription terminators within the operon, each of which is precede d by another stem-loop structure, the antiterminator, whose formation would prevent formation of the terminator stem-loop. These are located in the leader, the pyrR-pyrP intercistronic region, and the pyrP-pyrB intercistronic region. Northern (RNA) blot analysis has identified tr anscripts of lengths which coincide with termination at these proposed attenuation sites and whose relative abundances vary in the expected pyrimidine-dependent manner. Each antiterminator contains a 50-base co nserved sequence in its promoterproximal half. Various transcriptional fusions of the pyr promoter and surrounding sequences to promoterless reporter genes support an attenuation mechanism whereby when pyrimidi nes are abundant, the PyrR protein binds to the conserved sequence in the pyr mRNA and disrupts the antiterminator, permitting terminator ha irpin formation and promoting transcription termination. Deletion of p yrR from the chromosome resulted in the constitutive, elevated express ion 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 ur acil phosphoribosyltransferase activity. Analysis of pyrR and upp dele tion mutants demonstrated that upp, not pyrR, encodes the quantitative ly important uracil phosphoribosyltransferase activity. The pyrP gene probably encodes an integral membrane uracil permease.