Molecular recognition of pyr mRNA by the Bacillus subtilis attenuation regulatory protein PyrR.

The pyrimidine nucleotide biosynthesis (pyr) operon in Bacillus subtilis is regulated by transcriptional attenuation. The PyrR protein binds in a uridine nucleotide-dependent manner to three attenuation sites at the 5'-end of pyr mRNA. PyrR binds an RNA-binding loop, allowing a terminator hairpin to form and repressing the downstream genes. The binding of PyrR to defined RNA molecules was characterized by a gel mobility shift assay. Titration indicated that PyrR binds RNA in an equimolar ratio. PyrR bound more tightly to the binding loops from the second (BL2 RNA) and third (BL3 RNA) attenuation sites than to the binding loop from the first (BL1 RNA) attenuation site. PyrR bound BL2 RNA 4-5-fold tighter in the presence of saturating UMP or UDP and 150- fold tighter with saturating UTP, suggesting that UTP is the more important co-regulator. The minimal RNA that bound tightly to PyrR was 28 nt long. Thirty-one structural variants of BL2 RNA were tested for PyrR binding affinity. Two highly conserved regions of the RNA, the terminal loop and top of the upper stem and a purine-rich internal bulge and the base pairs below it, were crucial for tight binding. Conserved elements of RNA secondary structure were also required for tight binding. PyrR protected conserved areas of the binding loop in hydroxyl radical footprinting experiments. PyrR likely recognizes conserved RNA sequences, but only if they are properly positioned in the correct secondary structure.

Regulation of transcription of the Bacillus subtilis pyrG gene, encoding cytidine triphosphate synthetase.

The B. subtilis pyrG gene, which encodes CTP synthetase, is located far from the pyrimidine biosynthetic operon on the chromosome and is independently regulated. The pyrG promoter and 5' leader were fused to lacZ and integrated into the chromosomes of several B. subtilis strains having mutations in genes of pyrimidine biosynthesis and salvage. These mutations allowed the intracellular pools of cytidine and uridine nucleotides to be manipulated by the composition of the growth medium. These experiments indicated that pyrG expression is repressed by cytidine nucleotides but is largely independent of uridine nucleotides. The start of pyrG transcription was mapped by primer extension to a position 178 nucleotides upstream of the translation initiation codon. A factor-independent termination hairpin lying between the pyrG promoter and its coding region is essential for regulation of pyrG expression. Primer-extended transcripts were equally abundant in repressed and derepressed cells when the primer bound upstream of the terminator, but they were much less abundant in repressed cells when the primer bound downstream of the terminator. Furthermore, deletion of the terminator from pyrG-lacZ fusions integrated into the chromosome yielded elevated levels of expression that was not repressible by cytidine. We suggest that cytidine repression of pyrG expression is mediated by an antitermination mechanism in which antitermination by a putative trans-acting protein is reduced by elevated levels of cytidine nucleotides. Conservation of sequences and secondary structural elements in the pyrG 5' leaders of several other gram-positive bacteria indicates that their pyrG genes are regulated by a similar mechanism.

Purification and characterization of the DeoR repressor of Bacillus subtilis.

Transcription of the Bacillus subtilis dra-nupC-pdp operon is repressed by the DeoR repressor protein. The DeoR repressor with an N-terminal His tag was overproduced with a plasmid under control of a phage T5 promoter in Escherichia coli and was purified to near homogeneity by one affinity chromatography step. Gel filtration experimental results showed that native DeoR has a mass of 280 kDa and appears to exist as an octamer. Binding of DeoR to the operator DNA of the dra-nupC-pdp operon was characterized by using an electrophoretic gel mobility shift assay. An apparent dissociation constant of 22 nM was determined for binding of DeoR to operator DNA, and the binding curve indicated that the binding of DeoR to the operator DNA was cooperative. In the presence of low-molecular-weight effector deoxyribose-5-phosphate, the dissociation constant was higher than 1,280 nM. The dissociation constant remained unchanged in the presence of deoxyribose-1-phosphate. DNase I footprinting exhibited a protected region that extends over more than 43 bp, covering a palindrome together with a direct repeat to one half of the palindrome and the nucleotides between them.

Biochemical characterization of the heteromeric Bacillus subtilis dihydroorotate dehydrogenase and its isolated subunits.

Bacillus subtilis dihydroorotate dehydrogenase (DHOD) consists of two subunits, PyrDI (M(r) = 33,094) and PyrDII (M(r) = 28,099). The two subunits were overexpressed jointly and individually and purified. PyrDI was an FMN-containing flavoprotein with an apparent native molecular mass of 85,000. Overexpressed PyrDII formed inclusion bodies and was purified by refolding and reconstitution. Refolded PyrDII bound 1 mol FAD and 1 mol [2Fe-2S] per mol PyrDII. Coexpression and purification of PyrDI and PyrDII yielded a native holoenzyme complex with an apparent native molecular mass of 114,000 that indicated a heterotetramer (PyrDI(2)PyrDII(2)). The holoenzyme possessed dihydroorotate:NAD(+) oxidoreductase activity and could also reduce menadione and artificial dyes. Purified PyrDI also possessed DHOD activity but could not reduce NAD(+). Compared to PyrDI, the holoenzyme had a more than 20-fold smaller K(m) value for dihydroorotate, an approximately 50-fold smaller K(i) value for orotate, and approximately 500-fold greater catalytic efficiency. Dihydroorotate:NAD(+) oxidoreductase activity could be recovered by mixing the purified subunits. Recovered activity showed a clear dependence on FAD reconstitution of PyrDII but not on reconstitution with FeS clusters. PyrDII had a strong preference for FAD over FMN and bound it with an estimated K(d) value of 4.9 +/- 0.8 nM. pyrDII mutants containing alanine substitutions of the cysteine ligands to the [2Fe-2S] cluster failed to complement the pyr bradytrophy of a DeltapyrDII strain, indicating a requirement for the FeS cluster in PyrDII for normal function in vivo.

Regulation of the Bacillus subtilis pyrimidine biosynthetic operon by transcriptional attenuation: control of gene expression by an mRNA-binding protein.

The pyrimidine nucleotide biosynthetic (pyr) operon of Bacillus subtilis is regulated by a transcriptional attenuation mechanism in which termination of transcription at points upstream of the genes being regulated is promoted by the binding of a regulatory protein, PyrR, to specific sequences in the pyr mRNA. Binding of PyrR to pyr mRNA is stimulated by uridine nucleotides and causes changes in the mRNA secondary structure. This model is supported by extensive molecular genetic analysis. PyrR, which is encoded by the first gene of the pyr operon, is also a uracil phosphoribosyltransferase, although it has little amino acid sequence resemblance to other bacterial uracil phosphoribosyltransferases. Purified B. subtilis pyrR promotes attenuation of pyr transcription in vitro and binds specifically to pyr RNA sequences. The crystallographic structure of PyrR demonstrates the similarity of its tertiary structure to other phosphoribosyltransferases and suggests the surface to which RNA binds. PyrR is widely distributed among eubacteria and appears to regulate pyr genes not only by the attenuation mechanism found in B. subtilis, but also by a coupled transcription-translation attenuation mechanism and by acting as a translational repressor. PyrR illustrates the concept that transcriptional attenuation is a much more widespread and mechanistically versatile mechanism for the regulation of gene expression in bacteria than is generally recognized.

The Enterococcus faecalis pyr operon is regulated by autogenous transcriptional attenuation at a single site in the 5' leader.

The 5' end of the Enterococcus faecalis pyr operon specifies, in order, the promoter, a 5' untranslated leader, the pyrR 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 the pyrB 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-pyrB intercistronic regions. Several lines of evidence demonstrate that the E. 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.

Adaptation of an enzyme to regulatory function: structure of Bacillus subtilis PyrR, a pyr RNA-binding attenuation protein and uracil phosphoribosyltransferase.

BACKGROUND: The expression of pyrimidine nucleotide biosynthetic (pyr) genes in Bacillus subtilis is regulated by transcriptional attenuation. The PyrR attenuation protein binds to specific sites in pyr mRNA, allowing the formation of downstream terminator structures. UMP and 5-phosphoribosyl-1-pyrophosphate (PRPP), a nucleotide metabolite, are co-regulators with PyrR. The smallest RNA shown to bind tightly to PyrR is a 28-30 nucleotide stem-loop that contains a purine-rich bulge and a putative-GNRA tetraloop. PyrR is also a uracil phosphoribosyltransferase (UPRTase), although the relationship between enzymatic activity and RNA recognition is unclear, and the UPRTase activity of PyrR is not physiologically significant in B. subtilis. Elucidating the role of PyrR structural motifs in UMP-dependent RNA binding is an important step towards understanding the mechanism of pyr transcriptional attenuation. RESULTS: The 1.6 A crystal structure of B. subtilis PyrR has been determined by multiwavelength anomalous diffraction, using a Sm co-crystal. As expected, the structure of PyrR is homologous to those proteins of the large type I PRTase structural family; it is most similar to hypoxanthine-guanine-xanthine PRTase (HGXPRTase). The PyrR dimer differs from other PRTase dimers, suggesting it may have evolved specifically for RNA binding. A large, basic, surface at the dimer interface is an obvious RNA-binding site and uracil specificity is probably provided by hydrogen bonds from mainchain and sidechain atoms in the hood subdomain. These models of RNA and UMP binding are consistent with biological data. CONCLUSIONS: The B. subtilis protein PyrR has adapted the substrate- and product-binding capacities of a PRTase, probably an HGXPRTase, producing a new regulatory function in which the substrate and product are co-regulators of transcription termination. The structure is consistent with the idea that PyrR regulatory function is independent of catalytic activity, which is likely to be extremely low under physiological conditions.

Purification and characterization of Bacillus subtilis PyrR, a bifunctional pyr mRNA-binding attenuation protein/uracil phosphoribosyltransferase.

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 Km value for uracil is very pH-dependent; the Km for uracil at pH 7.7 is 990 +/- 114 muM, 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 Kd 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 muM. 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.

Mechanism of the synergistic end-product regulation of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase by nucleotides.

De novo purine nucleotide synthesis is regulated, at least in part, by end-product inhibition of glutamine PRPP amidotransferase. An important feature of this inhibition is the fact that certain synergistic nucleotide pairs give more than additive inhibition. The physiological importance of synergism is in amplifying regulation by the adenine and guanine nucleotide end products of de novo synthesis. Using a new method to quantitate synergism, ADP plus GMP were confirmed [Meyer, E., and Switzer, R. L. (1978) J. Biol. Chem. 254, 5397-5402] to give strong synergistic inhibition of Bacillus subtilis glutamine PRPP amidotransferase. An X-ray structure of the ternary enzyme.ADP.GMP complex established that ADP binds to the allosteric A site and GMP to the catalytic C site. GMP increased the binding affinity of ADP for the A site by approximately 20-fold. Synergism results from a specific nucleotide-nucleotide interaction that is dependent upon a nucleoside diphosphate in the A site and a nucleoside monophosphate in the C site. Furthermore, synergism is enhanced by the competition between nucleotide inhibitor and PRPP substrate for the C site. Purine base specificity results from a backbone carbonyl interaction of Lys305' with the 6-NH2 group of adenine in the A site and a Ser347 Ogamma interaction with the 2-NH2 group of guanine in the C site. Steric considerations favor binding of the nucleoside diphosphate to the A site. Site-directed replacements of key residues increased the nucleotide concentrations needed for 50% inhibition and in some cases perturbed synergism. Mutations in either of the nucleotide sites perturbed function at both sites, supporting the important role of synergism.

Transcriptional attenuation of the Bacillus subtilis pyr operon by the PyrR regulatory protein and uridine nucleotides in vitro.

Transcriptional attenuation of the pyrimidine biosynthetic (pyr) operon from Bacillus subtilis was reconstituted with an in vitro system that consisted of pyr DNA templates, B. subtilis RNA polymerase, four ribonucleoside triphosphates, and the purified B. subtilis PyrR regulatory protein. The templates used each specified one of the three known attenuation regions of the pyr operon. Runoff (read-though) and terminated transcripts of the predicted lengths were the only major products synthesized. Transcription of the template that specifies the 5' leader attenuation region of the operon was examined in detail. Termination of transcription at the attenuator was strongly promoted by the combination of PyrR plus UMP. The concentration of UMP required for half-maximal effect was 2.5 microM. UTP also promoted termination in the presence of PyrR, but concentrations 10-fold higher than UMP were required; UDP was only effective at 100 times the concentration of UMP. Other pyrimidine and purine metabolites tested did not affect termination. PRPP, which like UMP is a substrate for the uracil phosphoribosyltransferase activity of PyrR, antagonized UMP-dependent transcriptional termination, but uracil did not. Transcriptional attenuation by PyrR plus UMP was also demonstrated in vitro with templates from the other two pyr attenuation regions. The results strongly support the model for transcriptional regulation of the B. subtilis pyr operon previously proposed by R. J. Turner, Y. Lu, and R. L. Switzer (J. Bacteriol. 176:3708-3722, 1994).

Function of RNA secondary structures in transcriptional attenuation of the Bacillus subtilis pyr operon.

The Bacillus subtilis pyr operon is regulated by exogenous pyrimidines by a transcriptional attenuation mechanism. Transcription in vitro from pyr DNA templates specifying attenuation regions yielded terminated and read-through transcripts of the expected lengths. Addition of the PyrR regulatory protein plus UMP led to greatly increased termination. Synthetic antisense deoxyoligonucleotides were used to probe possible secondary structures in the pyr mRNA that were proposed to play roles in controlling attenuation. Oligonucleotides predicted to disrupt terminator structures suppressed termination, whereas oligonucleotides predicted to disrupt the stem of antiterminator stem-loops strongly promoted termination at the usual termination site. Oligonucleotides that disrupt a previously unrecognized stem-loop structure, called the anti-antiterminator, the formation of which interferes with formation of the downstream antiterminator, suppressed termination. We propose that transcriptional attenuation of the pyr operon is governed by switching between alternative antiterminator versus anti-antiterminator plus terminator structures, and that PyrR acts by UMP-dependent binding to and stabilization of the anti-antiterminator.

Evidence that the Bacillus subtilis pyrimidine regulatory protein PyrR acts by binding to pyr mRNA at three sites in vivo.

The Bacillus subtilis pyr operon is regulated by a transcriptional attenuation mechanism that requires the PyrR regulatory protein. Multicopy plasmids that could be transcribed to yield segments of RNA from the attenuation regions of the pyr operon induced derepression of chromosomal pyr genes, whereas plasmids that could not yield pyr RNA did not. We conclude that pyr RNA acts by titrating the PyrR protein and preventing it from regulating pyr attenuation.

Identification of a novel gene of pyrimidine nucleotide biosynthesis, pyrDII, that is required for dihydroorotate dehydrogenase activity in Bacillus subtilis.

An in-frame deletion in the coding region of a gene of previously unidentified function (which is called orf2 and which we propose to rename pyrDII) in the Bacillus subtilis pyr operon led to pyrimidine bradytrophy, markedly reduced dihydroorotate dehydrogenase activity, and derepressed levels of other enzymes of pyrimidine biosynthesis. The deletion mutation was not corrected by a plasmid encoding pyrDI, the previously identified gene encoding dihydroorotate dehydrogenase, but was complemented by a plasmid encoding pyrDII. We propose that pyrDII encodes a protein subunit of dihydroorotate dehydrogenase that catalyzes electron transfer from the pyrDI-encoded subunit to components of the electron transport chain.

Characterization of cis-acting mutations in the first attenuator region of the Bacillus subtilis pyr operon that are defective in pyrimidine-mediated regulation of expression.

A transcriptional attenuation mechanism for the regulation of pyr operon expression in Bacillus subtilis in which the PyrR regulatory protein binds pyr mRNA at three sites with similar sequences to cause transcription termination in response to elevated pyrimidine nucleotide pools has been proposed (R. J. Turner, Y. Lu, and R. L. Switzer, J. Bacteriol. 176:3708-3722, 1994). Twenty-seven mutants with cis-acting defects in the repression by pyrimidines of beta-galactosidase expression of a pyr-lacZ fusion-integrant were isolated as blue colonies on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) agar plates containing uracil and uridine after UV irradiation or treatment with mutagens or following mutD mutagenesis. These mutants showed normal repression of the chromosomal pyr operon by exogenous pyrimidines. Sequence analysis revealed 12 unique sites of mutation, which occurred in the conserved putative PyrR binding sequence (10 of the 12) or in the stem of the transcriptional terminator structure. These mutants strongly support the proposed model for regulation of the pyr operon.

Mutations in Bacillus subtilis PyrR, the pyr regulatory protein, with defects in regulation by pyrimidines.

The pyrimidine nucleotide biosynthetic (pyr) operon in Bacillus subtilis is regulated by a transcriptional attenuation mechanism in which PyrR, a bifunctional pyr RNA-binding attenuation protein/uracil phosphoribosyltransferase, plays a crucial role. A convenient procedure for isolation of pyrR mutants with defects in the regulation of pyr operon expression is described. The selection is based on the selection of spontaneous mutations that convert the pyrimidine-sensitive growth of cpa strain (lacking arginine-repressible carbamyl phosphate synthetase) to pyrimidine resistance. Twelve such mutants were isolated and sequenced. All resulted from point mutations in the pyrR gene.

The genetic and functional basis of purine nucleotide feedback-resistant phosphoribosylpyrophosphate synthetase superactivity.

The genetic and functional basis of phosphoribosylpyrophosphate synthetase (PRS) superactivity associated with purine nucleotide inhibitor-resistance was studied in six families with this X chromosome-linked purine metabolic and neurodevelopmental disorder. Cloning and sequencing of PRS1 and PRS2 cDNAs, derived from fibroblast total RNA of affected male patients by reverse transcription and PCR amplification, demonstrated that each PRS1 cDNA contained a distinctive single base substitution predicting a corresponding amino acid substitution in the PRS1 isoform. Overall, the array of substitutions encompassed a substantial portion of the translated sequence of PRS1 cDNA. Plasmid-mediated expression of variant PRS1 cDNAs in Escherichia coli BL21 (DE3/pLysS) yielded recombinant mutant PRS1s, which, in each case, displayed a pattern and magnitude of purine nucleoside diphosphate inhibitor-resistance comparable to that found in cells of the respective patient. Kinetic analysis of recombinant mutant PRS1s showed that widely dispersed point mutations in the X chromosome-linked PRPS1 gene encoding the PRS1 isoform result in alteration of the allosteric mechanisms regulating both enzyme inhibition by purine nucleotides and activation by inorganic phosphate. The functional consequences of these mutations provide a tenable basis for the enhanced production of phosphoribosylpyrophosphate, purine nucleotides, and uric acid that are the biochemical hallmarks of PRS superactivity.

Conformation of MgATP bound to 5-phospho-alpha-D-ribose 1-diphosphate synthetase by two-dimensional transferred nuclear Overhauser effect spectroscopy.

The conformation of MgATP bound at the active site of Salmonella typhimurium 5-phospho-alpha-D-ribose 1-diphosphate synthetase (PRibPP synthetase) has been investigated by two-dimensional transferred-NOE spectroscopy (TRNOESY). Inter-proton NOEs of the ligand were measured in the presence of the protein at several mixing times in the range of 40-300 ms at 500 MHz and 10 degrees C. Measurements were made at low ligand concentrations (approximately 1 mM) in order to avoid weak non-specific ligand-protein interactions and to ensure that the NOE arises from the ligand bound at the active site. The inter-proton distances were determined from the experimentally observed NOE buildup curves by comparing them with theoretical simulations obtained by using the complete relaxation matrix. These distances were used as constraints in molecular modeling and energy minimization calculations to deduce the structure of the bound ligand. PRibPP synthetase is known to appreciably aggregate so that it exists in multiple oligomeric forms in solution. The structure was determined under the assumption that the ligand assumes the same conformation on each subunit of every oligomer regardless of its size. On the basis of the rotational correlation time deduced for the enzyme-nucleotide complexes, it is estimated that the average oligomer of PRibPP synthetase, in the sample used for the TRNOESY measurements, consists of about 30 subunits, whereas the smallest active form of the protein is a pentamer. The conformation of enzyme-bound MgATP is described by a glycosidic torsion angle chi = 50 +/- 5 degrees and phase angle of pseudorotation P = 114.9 degrees corresponding to a 1T degree sugar pucker. It is noteworthy that the value of the glycosidic torsion angle obtained in this pyrophosphoryl transfer enzyme complex agrees well with those obtained previously for MgATP complexes of creatine kinase, pyruvate kinase (active and ancillary sites), and arginine kinase. The sugar pucker, on the other hand, differs from one enzyme complex to another.

Inhibition of human 5-phosphoribosyl-1-pyrophosphate synthetase by 4-amino-8-(beta-D-ribofuranosylamino)-pyrimido[5,4-d]pyrimidine-5'- monophosphate: evidence for interaction at the ADP allosteric site.

The kinetics of inhibition by the aminopyrimidopyrimidine nucleotide 4-amino-8-(beta-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine[-5' -monophosphate (APP-MP) were assessed with two human isozymes of 5-phosphoribosyl-1-pyrophosphate synthetase (PRS) (PRS1 and PRS2) and a mutant enzyme, S.M. PRS1, derived from an individual with PRS hyperactivity. In the presence of 1 mM potassium phosphate, APP-MP inhibited PRS1 and PRS2 with half-maximal inhibition (IC50) at 5.2 microM and 23.8 microM, respectively. The degree of inhibition for both enzymes was highly dependent on the phosphate concentration; IC50 values were 70 times higher in the presence of 50 mM potassium phosphate. APP-MP exhibited mixed noncompetitive-uncompetitive inhibition against PRS1, with a Kii value of 6.1 microM and a Kis value of 14.6 microM, and produced parabolic secondary plots of slope or intercept versus APP-MP concentration. In comparison, inhibition of PRS1 by ADP was of a mixed noncompetitive-competitive type, with a Kii value of 9.6 microM and a Kis value of 2.8 microM. A similar kinetic analysis was completed using S.M. PRS1, a mutant enzyme with a single amino acid substitution resulting in diminished sensitivity to feedback inhibition by nucleotides. The noncompetitive component of ADP inhibition of PRS1 was absent with S.M. PRS1 and ADP inhibition was purely competitive, with a Ki of 6.4 microM, APP-MP was a very poor inhibitor of S.M. PRS1, displaying uncompetitive characteristics and a Ki of 1.6 mM. These data indicate that APP-MP inhibits PRS1 with a strong element of noncompetitive inhibition and appears to interact specifically at the allosteric site used by ADP. These results contrast with those obtained with ADP, which has a strong component of ATP competitive inhibition and binds at the ATP site as well as at a second, allosteric, site.

Roles of the three transcriptional attenuators of the Bacillus subtilis pyrimidine biosynthetic operon in the regulation of its expression.

Expression of the Bacillus subtilis pyr operon is regulated by exogenous pyrimidines and the protein product of the first gene of the operon, PyrR. It has been proposed that PyrR mediates transcriptional attenuation at three untranslated segments of the operon (R.J. Turner, Y. Lu, and R.L. Switzer, J. Bacteriol., 176:3708-3722, 1994). In this study, transcriptional fusions of the pyr promoter followed by the pyr attenuation sequences, either individually or in tandem to a lacZ reporter gene, were used to examine the physiological functions of all three attenuators through their ability to affect beta-galactosidase expression. These fusions were studied as chromosomal integrants in various B. subtilis strains to examine the entire range of control by pyrimidines, PyrR dependence, amd developmental control of pyr gene expression. The nutritional regulation of each attenuator separately was roughly equivalent to that of the other two and was totally dependent upon PyrR, and that of tandem attenuators was cumulative. The regulation of a fusion of the spac promoter followed by the pyrP:pyrB intercistronic region to lacZ produced results similar to those obtained with the corresponding fusion containing the pyr promoter, demonstrating that attenuator-dependent regulation is independent of the promoter. Extreme pyrimidine starvation gave rise to two- to threefold-higher levels of expression of a pyr-lacZ fusion that lacked attenuators, independent of PyrR, than were obtained with cells that were not starved. Increased expression of a similar spac-lacZ fusion during pyrimidine starvation was also observed, however, indicating that attenuator-independent regulation is not a specific property of the pyr operon. Conversion of the initiator AUG codon in a small open reading frame in the pyrP:pyrB intercistronic region to UAG reduced expression by about half but did not alter regulation by pyrimidines, which excludes the possibility of a coupled transcription-translation attenuation mechanism. Developmental regulation of pyr expression during early stationary phase was found to be dependent upon the attenuators and PyrR, and the participation of SpoOA was excluded.