McNicholas P, Salavati R, Oliver D
Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, CT 06459, USA.
J Mol Biol. 1997 Jan 17;265(2):128-41. doi: 10.1006/jmbi.1996.0723.
The regulation of the Escherichia coli secA gene, whose translation is auto-repressed except when protein secretion becomes limiting, was investigated using a combination of genetic and biochemical approaches. Oligonucleotide-directed deletion and point mutagenesis was used to show that only the last quarter of the upstream gene, geneX, and the geneX-secA intergenic are essential for proper regulation. This region previously shown to contain a secretion-responsive element contains two predicted helices, helix I and II, the latter of which would occlude the secA Shine-Dalgarno sequence. Mutations that destabilized the lower portion of helix II increased secA basal expression, reduced auto-repression by SecA protein, but retained a normal pattern of derepression of secA expression during a protein export block. The introduction of compensatory mutations into helix II that were predicted to restore base-pairing restored secA regulation to wild-type levels or nearly so, suggesting that this helix does play a role in secA auto-regulation in vivo. In contrast, mutations in the lower portion of helix I decreased secA basal expression, reduced auto-repression by SecA protein, and abolished the responsiveness of secA expression to a protein export block. In this latter case introduction of compensatory mutations into helix I that were predicted to restore base-pairing did not restore proper secA regulation, indicating that specific nucleotides in this region are required for normal secA regulation. Primer-extension inhibition (toeprint) analysis with 30 S ribosoma subunits, tRNAMet, and a model segment of geneX-secA RNA carrying the relevant mutations was used to show that mutations that destabilized helix II increased ribosome binding at the secA translation initiation site, while mutations that perturbed helix I decreased ribosome binding at this site. Our results suggest strongly that there is a system of dual regulation of secA translation, whereby helix I serves as an activator element while helix II serves as a repressor element.
除蛋白质分泌受限的情况外，大肠杆菌secA基因的翻译受到自身抑制。我们采用遗传学和生物化学相结合的方法，对该基因的调控机制进行了研究。利用寡核苷酸定向缺失和点突变技术，结果表明，只有上游基因geneX的最后四分之一以及geneX-secA基因间区域对于正常调控至关重要。此前已证明该区域含有一个分泌反应元件，其中包含两个预测的螺旋结构，即螺旋I和螺旋II，后者会遮盖secA的Shine-Dalgarno序列。破坏螺旋II下部结构的突变会增加secA的基础表达，降低SecA蛋白的自身抑制作用，但在蛋白质输出受阻期间，secA表达的去抑制模式仍保持正常。在螺旋II中引入预计能恢复碱基配对的补偿性突变，可使secA调控恢复到野生型水平或接近野生型水平，这表明该螺旋在体内secA的自我调控中确实发挥了作用。相比之下，螺旋I下部的突变会降低secA的基础表达，减少SecA蛋白的自身抑制作用，并消除secA表达对蛋白质输出受阻的反应。在后一种情况下，在螺旋I中引入预计能恢复碱基配对的补偿性突变并不能恢复secA的正常调控，这表明该区域的特定核苷酸对于secA的正常调控是必需的。利用30S核糖体亚基、甲硫氨酰-tRNA以及携带相关突变的geneX-secA RNA模型片段进行引物延伸抑制（toeprint）分析，结果表明，破坏螺旋II的突变会增加核糖体在secA翻译起始位点的结合，而干扰螺旋I的突变会降低核糖体在该位点的结合。我们的结果有力地表明，存在一个secA翻译的双重调控系统，其中螺旋I作为激活元件，而螺旋II作为抑制元件。
