Investigation of the molecular interaction between pyr mRNA and the Bacillus subtilis attenuation regulatory protein, PyrR

In Bacillus subtilis, the operon encoding genes for de novo synthesis of pyrimidine nucleotides is regulated at three untranslated regions at the 5′ end of the operon. The PyrR protein binds a region of pyr mRNA called the “binding loop” in a uridine-nucleotide dependent manner, preventing formation of an antiterminator stem-loop structure and permitting terminator formation, aborting transcription at the three attenuation sites and repressing operon expression.; Titration with 5,5′-dithiobis-(2-nitrobenzoic acid) indicated that PyrR possessed 1 cysteine residue per monomer. Gel filtration chromatography suggested that PyrR alternated between dimeric and hexameric states of aggregation in a concentration-dependent manner. To study the interaction of PyrR and pyr mRNA, an electrophoretic gel mobility shift assays was developed. After assay optimization, binding was tight, specific, and responsive to uridine nucleotides. The binding loop from the second attenuation region (BL2) bound tightly to PyrR (K_d = 3 nM); low UMP concentrations caused 4-fold tighter binding; higher concentrations of UDP and UTP caused 5-fold and 150-fold tighter binding, respectively. PyrR bound the binding loop from the third attenuation region (BL3) 100-fold weaker than BL2, with a reduced response to uridine nucleotides. PyrR bound the binding loop from the first attenuation region (BL1) 10,000-fold weaker than BL2, with no response to uridine nucleotides.; Gel mobility shift experiments using progressively shorter variants of BL2 mRNA determined that the minimal RNA necessary for tight PyrR binding was 28 nt long. The stoichiometry of the PyrR-pyr mRNA interaction was determined to be equimolar using a gel mobility shift titration assay. The effects of 31 structural variants of BL2 on PyrR binding were studied. Twelve of the variations had little effect, three caused a moderate defect in binding, and sixteen severely disrupted binding. All PyrR-binding mRNAs share a conserved secondary structure, consisting of a lower stem, purine-rich internal bulge, upper stem, and terminal hexaloop, as well as two conserved sequence motifs. Variants that significantly altered the secondary structure of the mRNA disrupted binding. Variants that disrupted conserved sequences while leaving RNA secondary structure intact were tolerated in the upper stem, but disrupted binding in all other areas.