Abstract

The study of translation initiation in prokaryotes assumes that there should be a mechanism different from the canonical model, which postulates the formation of the pre-initiation complex through the interaction of the Shine-Dalgarno sequence (SD) at the 5′-end of mRNA and the anti-Shine-Dalgarno site at the 3′-end of 16S rRNA. In this paper we’ve studied the effect of TPS (Translation-initiation Promoting Site) on β-glucuronidase expression in E. coli cells at different cultivation temperatures. The examined leader sequences were cloned into the pET23c plasmid upstream the β-glucuronidase gene; protein expression was performed in E. coli BL21 (DE3) cells. β-glucuronidase activity was measured in bacterial cell extracts via paranitrophenyl b-d-glucuronide assay. The quantity of expressed protein was measured by Western blotting with following densitometry. It was shown that TPS increases the level of protein expression at stressful conditions (10 °C and 44 °C) 5–8 times compared to control. The combination of TPS and SD sites in the 5′-leader sequence of the mRNA created an enhancer that increased the expression level 2–3.6 times compared to a single SD-sequence. Based on the obtained data and the computer modeling of interaction between 16S rRNA and TPS, we proposed an alternative variation of prokaryotic translation initiation.

中文翻译：

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翻译促进位点（TPS）对大肠杆菌细胞中蛋白质表达的影响。

摘要

对原核生物翻译起始的研究假设应该有一个不同于经典模型的机制，该模型通过mRNA 5'端的Shine-Dalgarno序列（SD）的相互作用推测了起始前复合物的形成。以及位于16S rRNA 3'末端的反Shine-Dalgarno位点。在本文中，我们研究了不同培养温度下TPS（翻译起始促进位点）对大肠杆菌细胞中β-葡萄糖醛酸苷酶表达的影响。将检查的前导序列克隆到β-葡糖醛酸糖苷酶基因上游的pET23c质粒中。蛋白表达在大肠杆菌BL21（DE3）细胞中进行。通过对硝基苯基b- d测定细菌细胞提取物中的β-葡萄糖醛酸苷酶活性-葡糖苷酸测定。通过蛋白质印迹法和随后的光密度法测量表达的蛋白质的量。结果表明，与对照相比，TPS在应激条件下（10°C和44°C）可将蛋白质表达水平提高5至8倍。TPS和SD位点在mRNA的5'-前导序列中的结合产生了一个增强子，与单个SD序列相比，其表达水平提高了2–3.6倍。基于获得的数据和16S rRNA与TPS之间相互作用的计算机模型，我们提出了原核翻译起始的另一种变体。