This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The PyrR protein regulates expression of the genes in the de novo pyrimidine nucleotide biosynthesis (located in the pyr operon) in nearly all Gram-positive bacteria by a transcriptional attenuation mechanism. The PyrR protein binds in a uridine nucleotide dependent manner to binding loops at three attenuation sites at the 5 -end of pyr mRNA thereby allowing formation of a terminator hairpin. The affinity of PyrR for pyr mRNA is increased by uridine nucleotides, so an elevated pyrimidine level in the cell results in greater termination of transcription at sites upstream of the open reading frames of the pyr operon. The binding of Bacillus subtilis PyrR to defined RNA molecules was previously analyzed by electrophoretic gel mobility shift analysis to characterize the specificity of PyrR binding to pyr RNA. However, some artifacts of this method may have led to misleading conclusions. To test these findings and to further characterize the interaction between the protein and the RNA, we have thoroughly examined the binding of Bacillus caldolyticus PyrR to three RNA sequences by use of a filter binding assay. We also sought to characterize the PyrR-RNA interaction using fluorescence anisotropy. A 48 nucleotide RNA, for which the dissociation constant for PyrR binding was determined to be 0.1 nM by filter binding, was synthesized with a 5 -fluorescein tag (Dharmacon). Fluorescence anisotropy determinations indicated that the fluorescent tag was highly mobile in the RNA; the anisotropy was not altered by addition of excess PyrR. Fluorescence correlation spectroscopy also failed to detect evidence for the PyrR-RNA complex via changes in translational diffusion.
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