ABSTRACT
The 5' end of the Enterococcus faecalis pyr operon specifies, in order, the promoter, a 5' untranslated leader, the pyrR gene encoding the regulatory protein for the operon, a 39-nucleotide (nt) intercistronic region, the pyrP gene encoding a uracil permease, a 13-nt intercistronic region, and the pyrB gene encoding aspartate transcarbamylase. The 5' leader RNA is capable of forming stem-loop structures involved in attenuation control of the operon. No attenuation regions, such as those found in the Bacillus subtilis pyr operon, are present in the pyrR-pyrP or pyrP-pyrB intercistronic regions. Several lines of evidence demonstrate that the E. faecalis pyr operon is repressed by uracil via transcriptional attenuation at the single 5' leader termination site and that attenuation is mediated by the PyrR protein.
Subject(s)
5' Untranslated Regions , Bacterial Proteins , Enterococcus faecalis/genetics , Operon , Pentosyltransferases/genetics , Pyrimidine Nucleotides/biosynthesis , Repressor Proteins/genetics , Amino Acid Sequence , Base Sequence , Gene Expression Regulation, Bacterial , Genes, Bacterial , Molecular Sequence Data , Multigene Family , Nucleic Acid Conformation , Pentosyltransferases/biosynthesis , Recombinant Proteins/biosynthesis , Repressor Proteins/biosynthesis , Terminator Regions, Genetic , Transcription, Genetic , Uracil/pharmacologyABSTRACT
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.
