Brunel C, Romby P, Sacerdot C, de Smit M, Graffe M, Dondon J, van Duin J, Ehresmann B, Ehresmann C, Springer M
Institut de Biologie, Physico-Chimique, Paris, France.
J Mol Biol. 1995 Oct 20;253(2):277-90. doi: 10.1006/jmbi.1995.0552.
The expression of the gene encoding Escherichia coli threonyl-tRNA synthetase is negatively autoregulated at the translational level. The negative feedback is due to the binding of the synthetase to an operator site on its own mRNA located upstream of the initiation codon. The present work describes the characterisation of operator mutants that have the rare property of enhancing repression. These mutations cause (1) a low basal level of expression, (2) a temperature-dependent expression, and (3) an increased capacity of the synthetase to repress its own expression at low temperature. Surprisingly, this enhancement of repression is not explained by an increase of affinity of the mutant operators for the enzyme but by the formation, at low temperature, of a few supplementary base-pairs between the ribosomal binding site and a normally single-stranded domain of the operator. Although this additional base-pairing only slightly inhibits ribosome binding in the absence of repressor, simple thermodynamic considerations indicate that this is sufficient to increase repression. This increase is explained by the competition between the ribosome and repressor for overlapping regions of the mRNA. When the ribosomal binding site is base-paired, the ribosome cannot bind while the repressor can, giving the repressor the advantage in the competition. Thus, the existence of an open versus base-paired equilibrium in a ribosomal binding site of a translational operator amplifies the magnitude of control. This molecular amplification device might be an essential component of translational control considering the low free repressor/ribosome ratio of the low affinity of translational repressors for their target operators.
编码大肠杆菌苏氨酰 - tRNA合成酶的基因表达在翻译水平受到负向自动调节。负反馈是由于合成酶与其自身位于起始密码子上游的mRNA上的一个操纵基因位点结合所致。本研究描述了具有增强阻遏这一罕见特性的操纵基因突变体的特征。这些突变导致：（1）低基础表达水平；（2）温度依赖性表达；（3）合成酶在低温下抑制自身表达的能力增强。令人惊讶的是，这种阻遏增强并非由突变操纵基因对酶的亲和力增加所解释，而是由于在低温下核糖体结合位点与操纵基因一个通常为单链的结构域之间形成了一些额外的碱基对。尽管在没有阻遏物时这种额外的碱基配对仅轻微抑制核糖体结合，但简单的热力学考量表明这足以增加阻遏作用。这种增加是由核糖体和阻遏物对mRNA重叠区域的竞争来解释的。当核糖体结合位点碱基配对时，核糖体无法结合而阻遏物可以结合，从而使阻遏物在竞争中占优势。因此，翻译操纵基因的核糖体结合位点中开放与碱基配对平衡的存在放大了调控幅度。考虑到翻译阻遏物与其靶操纵基因的低亲和力以及低游离阻遏物/核糖体比率，这种分子放大机制可能是翻译调控的一个重要组成部分。
