BINDING OF PYRR ATTENUATION PROTEIN FROM BACILLUS CALDOLYTICUS TO PYR RNA

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。在几乎所有革兰氏阳性细菌中，PyrR蛋白通过转录衰减机制调节从头开始的嘧啶核苷酸生物合成(位于pyr操纵子)中的基因表达。PYRR蛋白以尿苷核苷酸依赖的方式与PYR基因5‘端三个衰减点的结合环结合，从而形成终止子发夹。尿苷核苷酸增加了PYR对PYR mRNA的亲和力，因此细胞内嘧啶水平的升高会导致PYR操纵子开放阅读框上游的转录终止程度更高。先前通过凝胶迁移率改变分析确定了枯草芽孢杆菌PyrR与确定的RNA分子的结合，以表征PyrR与pyr RNA结合的特异性。然而，这种方法的一些瑕疵可能导致了误导性的结论。为了检验这些发现并进一步表征蛋白质和RNA之间的相互作用，我们通过过滤结合试验彻底检测了Caldolyticus PyrR与三个RNA序列的结合。我们还试图用荧光各向异性来表征PyrR-RNA相互作用。用5？-荧光素标记(达哈拉康)合成了一个48个核苷酸的RNA，其与吡咯受体结合的解离常数为0.1 nm。荧光各向异性的测定表明，荧光标记在RNA中具有高度的移动性；过量的吡咯受体的加入不会改变这种各向异性。荧光相关光谱也未能通过平移扩散的变化检测到PyrR-RNA复合体的证据。

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.