Bacillus subtilis PyrR has been shown to mediate transcriptional attenuation at three separate sites within the pyrimidine nucleotide biosynthetic (pyr) operon. Molecular genetic evidence suggests that regulation is achieved by PyrR binding to pyr mRNA. PyrR is also a uracil phosphoribosyltransferase (UPRTase). Recombinant PyrR was expressed in Escherichia coli, purified to homogeneity, physically and chemically characterized, and examined with respect to both of these activities. Mass spectroscopic characterization of PyrR demonstrated a monomeric mass of 20,263 Da. Gel filtration chromatography showed the native mass of PyrR to be dependent on protein concentration and suggested a rapid equilibrium between dimeric and hexameric forms. The UPRTase activity of PyrR has a pH optimum of 8.2. The K m value for uracil is very pH-dependent; the K m for uracil at pH 7.7 is 990 ؎ 114 M, which is much higher than for most UPRTases and may account for the low physiological activity of PyrR as a UPRTase. Using an electrophoretic mobility shift assay, PyrR was shown to bind pyr RNA that includes sequences from its predicted binding site in the second attenuator region. Binding of PyrR to pyr RNA was specific and UMP-dependent with apparent K d values of 10 and 220 nM in the presence and absence of UMP, respectively. The concentration of UMP required for half-maximal stimulation of binding of PyrR to RNA was 6 M. The results support a model for the regulation of pyr transcription whereby termination is governed by the UMP-dependent binding of PyrR to pyr RNA and provide purified and characterized PyrR for detailed biochemical studies of RNA binding and transcriptional attenuation.The Bacillus subtilis pyrimidine biosynthetic (pyr) operon encodes all of the enzymes for the de novo biosynthesis of UMP and two additional cistrons encoding a uracil permease and the regulatory protein PyrR (1-4). On the basis of molecular genetic evidence it was proposed that PyrR regulates pyr expression through a transcriptional attenuation mechanism that acts at three separate sites within the operon, which are located in the 5Ј-untranslated leader, between the first (pyrR) and second (pyrP) genes, and between the second and third (pyrB) genes of the operon (3, 5). PyrR is proposed to regulate the ratio of terminated to readthrough transcripts at each attenuation site by permitting the formation of a -independent transcription terminator when exogenous pyrimidines are available. The binding of PyrR to pyr mRNA interferes with the formation of an alternative upstream stem-loop structure, the antiterminator, which is otherwise kinetically and thermodynamically favored. The presence of a conserved sequence within the 5Ј-stem of each antiterminator suggested a site within the pyr mRNA for interaction with PyrR (3); this site is the locus of several cis-acting mutants in the first pyr attenuator which are deficient in repression by pyrimidines (6).In addition, PyrR functions as a novel uracil phosphoribosyltransferase (UPRTase), 1 catalyzing the f...

PyrR is a protein that regulates the expression of genes and operons of pyrimidine nucleotide biosynthesis (pyr genes) in many bacteria. PyrR acts by binding to specific sequences on pyr mRNA and causing transcriptional attenuation when intracellular levels of uridine nucleotides are elevated. PyrR from Bacillus subtilis has been purified and extensively studied. In this work, we describe the purification to homogeneity and characterization of recombinant PyrR from the thermophile Bacillus caldolyticus and the crystal structures of unliganded PyrR and a PyrR-nucleotide complex. The B. caldolyticus pyrR gene was previously shown to restore normal regulation of the B. subtilis pyr operon in a pyrR deletion mutant. Like B. subtilis PyrR, B. caldolyticus PyrR catalyzes the uracil phosphoribosyltransferase reaction but with maximal activity at 60°C. Crystal structures of B. caldolyticus PyrR reveal a dimer similar to the B. subtilis PyrR dimer and, for the first time, binding sites for nucleotides. UMP and GMP, accompanied by Mg 2؉ , bind specifically to PyrR active sites. Nucleotide binding to PyrR is similar to other phosphoribosyltransferases, but Mg 2؉ binding differs. GMP binding was unexpected. The protein bound specific sequences of pyr RNA 100 to 1,000 times more tightly than B. subtilis PyrR, depending on the RNA tested and the assay method; uridine nucleotides enhanced RNA binding, but guanosine nucleotides antagonized it. The new findings of specific GMP binding and its antagonism of RNA binding suggest cross-regulation of the pyr operon by purines.

for PyrR that include a kinetically irreversible conformational change after binding of PRPP but before uracil binding were shown to account for the Ping Pong pattern of the enzyme. This mechanism was supported by the following experimental observations. The reverse reaction was extremely slow with a catalytic rate constant 3300 times smaller than for the forward reaction. Patterns of product inhibition of the forward reaction were consistent with a version of the irreversible conformational change model in which PyrR returns to the unliganded conformation before dissociation of UMP and were inconsistent with several other kinetic mechanisms. UMP and phosphoribosylpyrophosphate were shown by equilibrium dialysis to bind to free PyrR (dissociation constants of 27 ؎ 3 and 18 ؎ 2 M, respectively), but uracil and PP i did not bind at equilibrium concentrations up to 750 M. We propose that the conformational change kinetic model developed for PyrR can also account for numerous other reports of Ping Pong kinetics for various phosphoribosyltransferases that do not form the phosphoribosyl-enzyme intermediate predicted by classic Ping Pong kinetics.

The 5′ end of the Enterococcus faecalis pyr operon specifies, in order, the promoter, a 5′ untranslated leader, thepyrR gene encoding the regulatory protein for the operon, a 39-nucleotide (nt) intercistronic region, the pyrP gene encoding a uracil permease, a 13-nt intercistronic region, and thepyrB gene encoding aspartate transcarbamylase. The 5′ leader RNA is capable of forming stem-loop structures involved in attenuation control of the operon. No attenuation regions, such as those found in the Bacillus subtilis pyr operon, are present in the pyrR-pyrP or pyrP-pyrBintercistronic regions. Several lines of evidence demonstrate that theE. faecalis pyr operon is repressed by uracil via transcriptional attenuation at the single 5′ leader termination site and that attenuation is mediated by the PyrR protein.

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