Structure of the Nucleotide Complex of PyrR, the pyr Attenuation Protein from Bacillus caldolyticus , Suggests Dual Regulation by Pyrimidine and Purine Nucleotides

Abstract

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.

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

“…15). It is apparent that PurR and xanthine phosphoribosyltransferase are close relatives (366), that PyrR and hypoxanthine-guanine phosphoribosyltransferases are also closely related (337), and that they share ancestry with uracil phosphoribosyltransferases (336). As noted in Fig.…”

“…Altogether, the binding of PRPP and nucleotides to PyrR is similar to that of type I phosphoribosyltransferases. PyrR from B. caldolyticus has been proposed to exist as a dimer or tetramer (337,339,340), although B. subtilis PyrR has been shown to exist in a hexameric state (341). The dimerization pattern of PyrR is different from that of type I phosphoribosyltransferases, and the RNA-binding activity of PyrR has been proposed to be due to the exposure of a region of basic amino acid residues in the dimer.…”

“…Effectors of the quaternary structure are PRPP, uridine ribonucleotides, GTP, and pyr leader mRNA. A close resemblance of the crystal structure of GMP-bound B. caldolyticus PyrR to an evolutionarily distant hypoxanthine-guanine phosphoribosyltransferase suggests that the two proteins share ancestry (337). However, PyrR from both B. subtilis and B. caldolyticus have a low uracil phosphoribosyltransferase activity that can support slow growth of a pyrimidine requiring strain on uracil (248,342).…”

“…The three-dimensional structure of PyrR from three microbial sources has been published: B. subtilis (336), B. caldolyticus (337), and M. tuberculosis (338). The three-dimensional structure of T. thermophilus PyrR has also been solved (PDB code 1ufr).…”

Regulation of pyr Gene Expression in Mycobacterium smegmatis by PyrR-Dependent Translational Repression

“…The mechanism by which PyrR regulates the expression of pyr genes has been studied in Bacillus (17-19, 35, 36), Enterococcus (13), Lactococcus (20,21), and Lactobacillus (4,12,25) species. In these organisms PyrR acts by mediating a nucleotide-regulated transcription attenuation mechanism, which has been well characterized in genetic and biochemical studies with Bacillus subtilis (33) and Bacillus caldolyticus (8,14). PyrR binds to pyr mRNA at a site which lies upstream of the genes that it regulates.…”