Molecular recognition of pyr mRNA by the Bacillus subtilis attenuation regulatory protein PyrR
Molecular recognition of pyr mRNA by the Bacillus subtilis attenuation regulatory protein PyrR
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DOI：
10.1093/nar/29.23.4851
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发表时间：
2001-12-01
影响因子：
14.9
通讯作者：
Switzer, RL
中科院分区：
文献类型：
--
作者：
Bonner, ER;D'Elia, JN;Switzer, RL
枯草芽孢杆菌中的嘧啶核苷酸生物合成(PYR)操纵子受转录衰减的调节。PYR蛋白以尿苷核苷酸依赖的方式结合到PYR基因5‘端的三个衰减点。PyrR结合了一个RNA结合环，允许终止子发夹形成并抑制下游基因。凝胶迁移率改变分析表征了吡咯受体与特定RNA分子的结合。滴定结果表明，PyrR以等摩尔比与RNA结合。与第二个(BL2 RNA)和第三个(BL3 RNA)衰减点的结合环相比，PyrR与第一个(BL1 RNA)衰减点的结合环结合得更紧密。在饱和UMP或UDP存在下，PyrR结合的BL2 RNA紧密4-5倍，而在饱和UTP存在下，则紧密150倍，这表明UTP是更重要的协同调节因子。与PyrR紧密结合的最小RNA长度为28个核苷酸。对BL2 RNA的31个结构变体进行了PyrR结合亲和力测试。RNA的两个高度保守的区域，即上茎的末端环和顶部，以及富含嘌呤的内部凸起和下面的碱基对，对紧密结合至关重要。RNA二级结构的保守元件也是紧密结合所必需的。在羟基足迹实验中，PyrR保护了结合环的保守区。PyrR可能识别保守的RNA序列，但前提是它们位于正确的二级结构中。
The pyrimidine nucleotide biosynthesis (pyr) operon in Bacillus subtilis is regulated by transcriptional attenuation. The PyrR protein binds in a uridine nucleotide-dependent manner to three attenuation sites at the 5'-end of pyr mRNA. PyrR binds an RNA-binding loop, allowing a terminator hairpin to form and repressing the downstream genes. The binding of PyrR to defined RNA molecules was characterized by a gel mobility shift assay. Titration indicated that PyrR binds RNA in an equimolar ratio. PyrR bound more tightly to the binding loops from the second (BL2 RNA) and third (BL3 RNA) attenuation sites than to the binding loop from the first (BL1 RNA) attenuation site. PyrR bound BL2 RNA 4-5-fold tighter in the presence of saturating UMP or UDP and 150- fold tighter with saturating UTP, suggesting that UTP is the more important co-regulator. The minimal RNA that bound tightly to PyrR was 28 nt long. Thirty-one structural variants of BL2 RNA were tested for PyrR binding affinity. Two highly conserved regions of the RNA, the terminal loop and top of the upper stem and a purine-rich internal bulge and the base pairs below it, were crucial for tight binding. Conserved elements of RNA secondary structure were also required for tight binding. PyrR protected conserved areas of the binding loop in hydroxyl radical footprinting experiments. PyrR likely recognizes conserved RNA sequences, but only if they are properly positioned in the correct secondary structure.