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 inEscherichia 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. TheK m value for uracil is very pH-dependent; the K m for uracil at pH 7.7 is 990 ± 114 μm, 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 K d 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 μm. The results support a model for the regulation ofpyr 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.

• Publication year

  1998

• Venue

  Journal of Biological Chemistry

• Publication date

  1998-03-06

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1074/jbc.273.10.5932PMID 9488732
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• PyrR bound pyr RNA specifically and in a UMP-dependent manner, and the results support a model in which UMP-dependent PyrR binding helps govern transcriptional attenuation in the pyr operon.

  Confidence 0.96

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewKiller Whale (322360f1c1) review
• PyrR displayed uracil phosphoribosyltransferase activity with a pH optimum of 8.2 and a very pH-dependent Km for uracil, reaching 990 ± 114 μM at pH 7.7.

  Confidence 0.97

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewKiller Whale (322360f1c1) review
• Mass spectrometry and gel filtration chromatography indicated a 20,263 Da monomer and a concentration-dependent equilibrium between native dimeric and hexameric forms of PyrR.

  Confidence 0.95

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewKiller Whale (322360f1c1) review
• Recombinant Bacillus subtilis PyrR was expressed in Escherichia coli and purified to homogeneity for biochemical characterization.

  Confidence 0.98

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewKiller Whale (322360f1c1) review

• bacillus subtilis pyrr

  protein

  A B. subtilis bifunctional protein studied here for RNA binding and uracil phosphoribosyltransferase activity.

  Aliases: PyrR

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewKiller Whale (322360f1c1) review
• gel filtration chromatography

  analytical method

  A size-based chromatography method used here to assess the native mass of PyrR.

  Aliases: size-exclusion chromatography, gel filtration

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewKiller Whale (322360f1c1) review
• km for uracil

  kinetic parameter

  The Michaelis constant describing PyrR's affinity for uracil as a substrate.

  Aliases: uracil Km, K m for uracil

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewKiller Whale (322360f1c1) review
• mass spectrometry

  analytical method

  An analytical technique used here to determine the monomeric mass of PyrR.

  Aliases: mass spectroscopic characterization

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewKiller Whale (322360f1c1) review
• oligomeric state

  structural state

  The native multimeric form adopted by PyrR under different protein concentrations.

  Aliases: native quaternary state

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewKiller Whale (322360f1c1) review
• ph optimum

  parameter

  The pH value at which PyrR's uracil phosphoribosyltransferase activity is maximal.

  Aliases: optimal pH

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewKiller Whale (322360f1c1) review
• purification to homogeneity

  protein preparation

  Isolation of PyrR as a highly pure protein preparation suitable for characterization and assays.

  Aliases: purified to homogeneity

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewKiller Whale (322360f1c1) review
• pyr operon

  gene cluster

  The Bacillus subtilis pyrimidine nucleotide biosynthetic operon controlled by PyrR.

  Aliases: pyrimidine biosynthetic operon

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewKiller Whale (322360f1c1) review
• pyr rna

  RNA

  RNA from the pyr operon that contains the predicted PyrR binding site in the second attenuator region.

  Aliases: pyr mRNA

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewKiller Whale (322360f1c1) review
• recombinant protein expression

  method

  Production of PyrR from a recombinant construct in Escherichia coli.

  Aliases: recombinant expression, expression in E. coli

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewKiller Whale (322360f1c1) review
• transcriptional attenuation

  regulatory mechanism

  A regulatory mechanism in which RNA structure and protein binding influence transcription termination.

  Aliases: attenuation

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewKiller Whale (322360f1c1) review
• ump

  nucleotide

  Uridine monophosphate, the nucleotide effector that modulates PyrR-RNA binding in the assays.

  Aliases: uridine monophosphate

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewKiller Whale (322360f1c1) review
• ump-dependent rna binding

  binding interaction

  Binding of PyrR to pyr RNA whose strength is modulated by UMP.

  Aliases: UMP-dependent binding

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewKiller Whale (322360f1c1) review
• uracil phosphoribosyltransferase

  enzyme

  An enzyme that converts uracil into UMP in pyrimidine salvage metabolism.

  Aliases: UPRTase

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewKiller Whale (322360f1c1) review

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