m RNA5′端不同位置的二级结构对原核生物翻译起始的影响

Effect of Secondary Structure in Different Position of the 5′End of mRNA on the Translation Initiation in Escherichia coli

一个 2 1 bp的序列插入 B50中的人 PCNA- Lac Z′融合蛋白 m RNA的 SD序列的上游 1 1 bpH ind 切点处 ,构成正、反向插入的两个重组质粒 B50 il、B50 i2 .通过计算机程序对 m RNA二级结构的模拟分析 ,B50 il的插入序列在翻译起始区 (TIR)前形成一个发夹结构 ,但不影响 TIR的二级结构 ;B50 i2的插入序列在 TIR 5′端形成一个二级结构 ;而另一克隆 D1 3与 B50因 SD前后的 6个和 7个碱基序列的不同 ;使翻译起始区 TIR的 SD到 AUG附近的二级结构不相同 .实验测定的四者的β-半乳糖苷酶活性与计算机计算的解开 m RNA TIR的二级结构所需能量ΔE进行比较 ,其结果说明 m RNA TIR前面的二级结构对翻译起始无影响 ,而位于 TIR的 5′端的二级结构对翻译起始效率是有影响的 .不过 ,它与 m RNA TIR的 SD到 AUG的附近的二级结构对翻译起始效率的影响相比 ,TIR5′端二级结构的影响比较小 .同时 ,PCNA- Lac Z和 PCNA- Lac Z′两种融合蛋白的β-半乳糖苷酶活性也进行了比较 ,酶活性有差别 ,但不同质粒酶活性的比值仍相同 .

A 21 bp fragment was inserted into the Hin d Ⅲ site,11 bp upstream of the Shine/Dalgarno(SD)sequence,of the plasmid B50 which was a clone for the expression of the human PCNA Lac Z′ gene.If the fragment was inserted in forward direction,the plasmid B50il was obtained and computer analysis revealed that a hairpin formed ahead of the translation initiation region(TIR)but not affected the TIR.If it was inserted inversely,B50i2 was obtained,with a secondary structure at the 5′ part of the TIR.Another plasmid,D13,was different from B50 in the secondary structure of the TIR,because of different 6 bp upstream and 7 bp downstream of the SD sequence.By the comparison of the β galactosidase activities and the energies( ΔE ) required for releasing the secondary structure of the TIR calculated by the computer,of the four plasmids,it indicates that the efficiency of translation initiation is not affected by the secondary structure ahead of the TIR,but by that of the 5′ part TIR,though less than that near SD and AUG.Meanwhile,the expression of the Lac Z was measured and compared with that of the Lac Z′ .The β galactosidase activity with holoenzyme was a little bit higher than the α peptide of enzyme,but the ratios among the different plasmids were the same.