TRANSLATIONAL REGULATION OF THE SPC OPERON IN ESCHERICHIA-COLI - IDENTIFICATION AND STRUCTURAL-ANALYSIS OF THE TARGET SITE FOR S8 REPRESSOR PROTEIN
TRANSLATIONAL REGULATION OF THE SPC OPERON IN ESCHERICHIA-COLI - IDENTIFICATION AND STRUCTURAL-ANALYSIS OF THE TARGET SITE FOR S8 REPRESSOR PROTEIN
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DOI：
10.1016/0022-2836(88)90578-5
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发表时间：
1988-11-20
影响因子：
5.6
通讯作者：
NOMURA, M
中科院分区：
文献类型：
--
作者：
CERRETTI, DP;MATTHEAKIS, LC;NOMURA, M
大肠杆菌的 spc 核糖体蛋白操纵子受到核糖体蛋白 S8（一种翻译阻遏蛋白）的反馈调节。我们分析了负责这种调节的 spc mRNA 区域。首先，我们确定 mRNA 上的 S8 靶位点靠近操纵子中编码核糖体蛋白 L5 的第三个基因的翻译起始位点。这是通过构建携带 lac 转录控制下的 spc 操纵子核糖体蛋白基因及其缺失衍生物的杂合质粒，并进行体内和体外蛋白质合成实验来完成的。接下来，通过使用结构特异性核酸酶分析从噬菌体 SP6 启动子合成的 5'' 末端标记的 RNA，研究了该区域的二级结构。借助计算机预测RNA折叠，推导出与结果一致的二级结构模型。此外，我们对鼠伤寒沙门氏菌、普通变形杆菌和粘质沙雷氏菌的相应区域进行了克隆和测序，发现了五个“补偿”取代，支持一些推导的 mRNA 螺旋结构。没有任何碱基变化与推导的二级结构模型不一致。最后，定点诱变实验已经确定了对于调节重要的碱基，包括代表两个螺旋区域中的每一个的两个碱基配对位点。由此得出的结论是，位于这两个螺旋区之间的一些特定核苷酸残基直接参与S8识别，并且这两个螺旋区的功能是维持这些核苷酸残基的正确方向。将 spc mRNA 上的 S8 靶位点的结构与 rRNA 上已知的 S8 结合位点的结构进行比较，发现一级和二级结构具有惊人的相似性。特别是，发现该区域中远缘相关细菌物种之间保守的 rRNA 一级序列与 mRNA 中相应位置的序列相同。这些结果表明，S8 阻遏蛋白的相同结构特征参与了与 16 S rRNA 和 mRNA 靶位点的相互作用。
The spc ribosomal protein operon of Escherichia coli is feedback-regulated by ribosomal protein S8, a translational repressor. We have analyzed the region of the spc mRNA that is responsible for this regulation. First, we have established that the S8 target site on the mRNA is near the translation start site of the third gene encoding ribosomal protein L5 in the operon. This was done by constructing hybrid plasmids carrying spc operon ribosomal protein genes under lac transcriptional control, as well as their deletion derivatives, and carrying out both in vivo and in vitro protein synthesis experiments. Next, the secondary structure of this region was studied by analyzing 5'' end-labeled RNA synthesized from the phage SP6 promoter using structure-specific nucleases. A secondary structure model consistent with the results was deduced with the aid of a computer prediction of RNA folding. In addition, we cloned and sequenced the corresponding region from Salmonella typhimurium, Proteus vulgaris and Serratia marcescens and found five "compensating" substitutions that support some of the deduced helical structures of mRNA. None of the base changes was inconsistent with the deduced secondary structure model. Finally, site-directed mutagenesis experiments have identified bases important for regulation, including two base-paired sites representing each of two helical regions. This has led to the conclusion that some specific nucleotide residues located between these two helical regions are directly involved in S8 recognition, and that the function of the two helical regions is to maintain the proper orientation of these nucleotide reisudes. Comparison of the structure of the S8 target site on the spc mRNA with the known S8 binding site on rRNA has revealed a striking similarity in both primary and secondary structures. In particular, primary sequences of rRNA conserved among distantly related bacterial species in this region is found to be identical with the sequences at the corresponding positions in mRNA. These results suggest that the same structural features of the S8 repressor protein are involved in the interaction with both 16 S rRNA and the mRNA target site.