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 attenuatio n, The PyrR attenuation protein binds to specific sites in pyr mRNA, a llowing the formation of downstream terminator structures. UMP and 5-p hosphoribosyl-1-pyrophosphate (PRPP), a nucleotide metabolite, are co- regulators with PyrR. The smallest RNA shown to bind tightly to PyrR i s a 28-30 nucleotide stem-loop that contains a purine-rich bulge and a putative-GNRA tetraloop. PyrR is also a uracil phosphoribosyltransfer ase (UPRTase), although the relationship between enzymatic activity an d RNA recognition is unclear, and the UPRTase activity of PyrR is not physiologically significant in B. subtilis, Elucidating the role of Py rR structural motifs in UMP-dependent RNA binding is an important step towards understanding the mechanism of pyr transcriptional attenuatio n. Results: The 1.6 Angstrom 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 simi lar to hypoxanthine-guanine-xanthine PRTase (HGXPRTase), The PyrR dime r differs from other PRTase dimers, suggesting it may have evolved spe cifically for RNA binding. A large, basic, surface at the dimer interf ace is an obvious RNA-binding site and uracil specificity is probably provided by hydrogen bonds from mainchain and sidechain atoms in the h ood subdomain. These models of RNA and UMP binding are consistent with biological data. Conclusions: The B. subtilis protein PyrR has adapte d the substrate-and product-binding capacities of a PRTase, probably a n HGXPRTase, producing a new regulatory function in which the substrat e and product are cc-regulators of transcription termination, The stru cture is consistent with the idea that PyrR regulatory function is ind ependent of catalytic activity, which is likely to be extremely low un der physiological conditions.