Portier C, Philippe C, Dondon L, Grunberg-Manago M, Ebel J P, Ehresmann B, Ehresmann C
Institut de Biologie Physico-chimique, Paris, France.
Biochim Biophys Acta. 1990 Aug 27;1050(1-3):328-36. doi: 10.1016/0167-4781(90)90190-d.
The expression of ribosomal protein S15 is shown to be translationally and negatively autocontrolled using a fusion within a reporter gene. Isolation and characterization of several deregulated mutants indicate that the regulatory site (the translational operator site) overlaps the ribosome loading site of the S15 messenger. In this region, three domains, each exhibiting a stem-loop structure, were determined using chemical and enzymatic probes. The most downstream hairpin carries the Shine-Dalgarno sequence and the initiation codon. Genetic and structural data derived from mutants constructed by site-directed mutagenesis show that the operator is a dynamic structure, two domains of which can form a pseudoknot. Binding of S15 to these two domains suggests that the pseudoknot could be stabilized by S15. A model is presented in which two alternative structures would explain the molecular basis of the S15 autocontrol.
利用报告基因内的融合体，核糖体蛋白S15的表达被证明受到翻译水平的负向自我调控。几个失调突变体的分离和表征表明，调控位点（翻译操纵子位点）与S15信使核糖核酸的核糖体装载位点重叠。在该区域，使用化学和酶促探针确定了三个均呈现茎环结构的结构域。最下游的发夹结构携带Shine-Dalgarno序列和起始密码子。通过定点诱变构建的突变体的遗传和结构数据表明，操纵子是一种动态结构，其中两个结构域可形成假结。S15与这两个结构域的结合表明，假结可被S15稳定。本文提出了一个模型，其中两种替代结构可解释S15自我调控的分子基础。
