Regulation of the Bacillus subtilis pyrimidine biosynthetic (pyr) gene cluster by an autogenous transcriptional attenuation mechanism

Abstract

A complete transcript of the Bacillus subtilis pyr operon contains the following elements in 5' to 3' order: a 151-nucleotide (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; and eight overlapping cistrons encoding all of the six enzymes for de novo pyrimidine biosynthesis. Transcription is controlled by the availability of pyrimidines via an attenuation mechanism. There are three 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, the pyrR-pyrP intercistronic region, and the pyrP-pyrB intercistronic region. Northern (RNA) blot analysis has identified transcripts 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 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. 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 activity. Analysis of pyrR and upp deletion mutants demonstrated that upp, not pyrR, encodes the quantitatively important uracil phosphoribosyltransferase activity. The pyrP gene probably encodes an integral membrane uracil permease.

Mutations affecting transcription pausing in the Bacillus subtilis pyr operon

“…Construction and integration of pyr-lacZ fusion-integrant strains and assays of lacZ regulation by pyrimidines Excision of the pyr DNA fragments with EcoRI and BamHI endonucleases from the plasmids described above in which the mutations were constructed, followed by ligation of the EcoRIBamH1 pyr fragments into the pDH32 plasmid (Grandoni et al 1993) yielded plasmids in which the pyr promoter and native or mutated attenuation regions were placed upstream of a lacZ reporter gene as transcriptional fusions. The corresponding plasmids were integrated into the amyE locus of three B. subtilis strains: a wild type strain (MO199 trpC2 pheA amyE::erm, Grandoni et al 1993), a constitutive ΔpyrR strain (DB104 his, nprR2 nprE18 ΔnprA3 ΔpyrR, Turner et al 1994), and a pyrimidine auxotroph HC11 (his, nprR2 nprE18 ΔnprA3 pyrB::Spec r , Hu and Switzer 1995) as previously described (Lu et al 1995).…”

Two genes encoding uracil phosphoribosyltransferase are present in Bacillus subtilis

“…The incorporation rates presented in Table 2 were determined as previously described (5). The uracil incorporation data confirms the re- (2), and pyrR-Bacillus subtilis (PyrR) (17). Shaded residues represent amino acids which are identical among at least three of the five UPRTases.…”

“…pJAM200 was shown to contain a sequence identical to the 181-amino-acid open reading frame (PyrR) in the pyr operon shown to be the regulator of the structural genes responsible for the de novo synthesis of UMP (17). PyrR is thus an example of a regulatory protein having catalytic activity.…”

“…An isogenic series of strains containing either the ⌬pyrR mutation, the upp-11 mutation, or both mutations was constructed in the following way. Strains DB104 (hisA1) and DB104⌬pyrR (hisA1 ⌬pyrR) (17) were transduced with AR9 phages propagated on strain HH222, thus producing the strains HH207 (hisA1 upp-11) and HH211 (hisA1 ⌬pyrR upp-11), respectively. The effect of the upp and ⌬pyrR mutations and the upp ⌬pyrR double mutation was analyzed in four ways: (i) assay of UPRTase activity in crude extract, (ii) pyrimidine excretion, (iii) effect of the pyrimidine analog 5-fluorouracil on growth, and (iv) incorporation of uracil and uridine.…”

Antitermination of transcription of catabolic operons

“…The antiterminator is a stem-loop structure which overlaps the terminator by four bp and prevents its formation. The B. subtilis pur and pyr operons seem to be regulated by a similar mechanism (Zalkin and Ebbole, 1988;Turner et al, 1994).…”