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Novel RNA-Binding Proteins Isolation by the RaPID Methodology
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Novel RNA-Binding Proteins Isolation by the RaPID Methodology

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Molecular recognition of pyr mRNA by the Bacillus subtilis attenuation regulatory protein PyrR

E R Bonner1, J N D'Elia, B K Billips

  • 1Department of Biochemistry, University of Illinois, 600 South Mathews Avenue, Urbana, IL 61801, USA.

Nucleic Acids Research
|December 1, 2001
PubMed

Insights

The PyrR protein regulates gene expression in Bacillus subtilis by binding to specific RNA structures. Uridine triphosphate (UTP) significantly enhances PyrR binding affinity to these regulatory RNA elements.

Area of Science:

  • Molecular Biology
  • Gene Regulation
  • Biochemistry

Background:

  • The pyrimidine nucleotide biosynthesis (pyr) operon in Bacillus subtilis is controlled by transcriptional attenuation.
  • The PyrR protein is a key regulator that binds to attenuation sites on pyr mRNA.
  • PyrR binding to RNA is dependent on uridine nucleotides, leading to gene repression.

Purpose of the Study:

  • To characterize the binding interaction between the PyrR protein and its RNA targets.
  • To identify the specific RNA structural elements and nucleotide co-regulators crucial for PyrR binding.
  • To elucidate the mechanism of transcriptional attenuation mediated by PyrR.

Main Methods:

  • Gel mobility shift assays were used to study PyrR-RNA binding kinetics and affinity.
  • Hydroxyl radical footprinting experiments were performed to identify protected RNA regions.
  • Systematic structural variants of attenuation site RNA were synthesized and tested for binding.

Main Results:

  • PyrR binds RNA in an equimolar ratio, with higher affinity for BL2 and BL3 RNA compared to BL1 RNA.
  • Uridine triphosphate (UTP) significantly increased PyrR binding affinity (up to 150-fold), indicating its role as a co-regulator.
  • A minimal RNA length of 28 nucleotides was required for tight binding, with conserved RNA sequence and secondary structure elements being critical.

Conclusions:

  • PyrR recognizes specific conserved RNA sequences within a defined secondary structure for efficient binding.
  • UTP is a critical co-regulator that enhances PyrR binding to attenuation sites, thereby controlling pyr operon expression.
  • The study provides insights into the molecular mechanisms of transcriptional attenuation and RNA-protein interactions in Bacillus subtilis.

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