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.
Turner
R J
RJ
Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, USA.
Bonner
E R
ER
Grabner
G K
GK
Switzer
R L
RL
D001412:Bacillus subtilis / Q000737:chemistry* / Q000201:enzymology; D001426:Bacterial Proteins; D001483:Base Sequence; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010430:Pentosyltransferases / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011743:Pyrimidines / Q000096:biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012097:Repressor Proteins / Q000737:chemistry*; D014158:Transcription, Genetic / Q000235:genetics; D014542:Uridine Monophosphate / Q000494:pharmacology
D001426:Bacterial Proteins; D011743:Pyrimidines; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014542:Uridine Monophosphate; D010430:Pentosyltransferases; C098851:PyrR protein, bacteria; C023269:uracil phosphoribosyltransferase