PURIFICATION AND CHARACTERIZATION OF BACILLUS-SUBTILIS PYRR, A BIFUNCTIONAL PYR MESSENGER-RNA-BINDING ATTENUATION PROTEIN URACIL PHOSPHORIBOSYLTRANSFERASE/

Bacillus subtilis PyrR Has been shown to mediate transcriptional atten uation at three separate sites within the pyrimidine nucleotide biosyn thetic (pyr) operon, Molecular genetic evidence suggests that regulati on is achieved by PyrR binding to pyr mRNA, PyrR is also a uracil phos phoribosyltransferase (UPRTase), Recombinant PyrR was expressed in Esc herichia coli, purified to homogeneity, physically and chemically char acterized, and examined with respect to both of these activities. Mass spectroscopic characterization of PyrR demonstrated a monomeric mass of 20,263 Da. Cel filtration chromatography showed the native mass of PyrR to be dependent on protein concentration and suggested a rapid eq uilibrium 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-dep endent; the K-m for uracil, at pH 7.7 is 990 +/- 114 mu M, which is mu ch. higher than far most UPRTases and may account for the low physiolo gical activity of PyrR as a UPRTase, Using an electrophoretic mobility shift assay, PyrR was shown to bind pyr RNA that includes sequences f rom its predicted binding site in the second attenuator region, Bindin g 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, respective ly. The concentration of UMP required for half-maximal stimulation of binding of PyrR. to RNA was 6 mu M, The results support a model for th e regulation of pyr transcription whereby termination is governed by t he UMP-dependent binding of PyrR to pyr RNA and provide purified and c haracterized PyrR for detailed biochemical studies of RNA binding and transcriptional attenuation.