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

Preethi Chander,¹ Kari M. Halbig,^2, Jamie K. Miller,^2, Christopher J. Fields,² Heather K. S. Bonner,^2, Gail K. Grabner,^2,¶ Robert L. Switzer,² and Janet L. Smith^1*

Department of Biological Sciences, Purdue University, West Lafayette, Indiana,¹ Department of Biochemistry, University of Illinois, Urbana, Illinois²

Received 6 October 2004/ Accepted 17 November 2004

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 Mg²⁺, bind specifically to PyrR active sites. Nucleotide binding to PyrR is similar to other phosphoribosyltransferases, but Mg²⁺ 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.

Present address: Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2128.

Present address: UC Davis Cancer Center, Sacramento, CA 95817.

Present address: Danforth Plant Science Center, St. Louis, MO 63132.

^¶ Present address: Department of Microbiology, University of Illinois, Urbana, IL 61801.

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