Purification and Characterization of Bacillus subtilis PyrR, a Bifunctional pyr mRNA-binding Attenuation Protein/Uracil Phosphoribosyltransferase

Abstract

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...

Bacillus subtilis glutamine synthetase regulates its own synthesis by acting as a chaperone to stabilize GlnR–DNA complexes

“…This regulation can occur by a variety of different mechanisms. For instance, aconitase and uracil phosphoribosyltransferase are regulatory enzymes that bind RNA (29,30). Proline oxidase and biotin synthetase contain separate DNAbinding domains that allow these proteins to function as transcriptional repressors (31,32).…”

Kinetic Studies of the Uracil Phosphoribosyltransferase Reaction Catalyzed by the Bacillus subtilis Pyrimidine Attenuation Regulatory Protein PyrR

“…This finding was unexpected because the deduced amino acid sequences of PyrR proteins from various bacteria bear no significant sequence similarity outside of a short sequence in the active site to the sequences of previously characterized bacterial UPRTases, which are encoded by upp genes (1). Nonetheless, purified B. subtilis PyrR, when assayed under optimal conditions, has a UPRTase-specific activity comparable with purified bacterial upp-encoded UPRTases (8). Furthermore, the three-dimensional structure of B. subtilis PyrR, which was solved at high resolution by Tomchick et al (9), demonstrated that the PyrR structure is very similar to other Type I phosphoribosyltransferases (10).…”

“…This was the case throughout the pH range from 7.2 to 9.7 and at all substrate concentrations studied. Values for the maximal velocity and Michaelis constants for PRPP and uracil from pH 7.7 to 9.7 have been previously published (8). A Ping Pong kinetic pattern is characteristic of enzymes that form a covalent intermediate with a portion of a substrate molecule, which in the case of UPRTase would indicate the following mechanism.…”

“…Assays for UPRTase and Exchange Reactions-The UPRTase forward (uracil ϩ PRPP 3 PP i ϩ UMP) reaction was assayed by Method 2 previously described (8) Equilibrium Dialysis Studies of Ligand Binding by PyrR-The procedure described by Gibson et al (14) was used for equilibrium dialysis, except that ultrathin regenerated cellulose circular (1 cm diameter) dialysis membranes (10,000 molecular weight cut-off, The Nest Group, Southborough, MA) were used. Dialysis was in 100 mM Tris acetate, 10 mM potassium acetate, 20% glycerol, pH 7.5, at 0°C for 40 -48 h. Equimolar MgCl 2 was included when uracil, UMP, and UTP are ligands; 3-and 2-fold molar excess MgCl 2 were added with PRPP and PP i , respectively.…”

“…Materials-B. subtilis PyrR was purified by the procedure of Turner et al (8) with the following modifications. The supernatant fluid from the streptomycin precipitation step was applied directly to the QAE-* The costs of publication of this article were defrayed in part by the payment of page charges.…”