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首页> 外文期刊>Nucleic acids research >An unstructured mRNA region and a 5′ hairpin represent important elements of the E.coli translation initiation signal determined by using the bacteriophage 17 gene 1 translation start site
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An unstructured mRNA region and a 5′ hairpin represent important elements of the E.coli translation initiation signal determined by using the bacteriophage 17 gene 1 translation start site

机译：非结构化的mRNA区域和5'发夹代表使用噬菌体17基因1翻译起始位点确定的大肠杆菌翻译起始信号的重要元素

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Gene 1 of bacteriophage T7 early region-the RNA polymerase gene-is very actively translated during the infectious cycle of this phage. A 29 base pair fragment of its ribosome binding site containing the initiation triplet, the Shine-Dalgarno sequence (S-D), 10 nucleotides (nt) upstream and 6 nt downstream of these central elements was cloned into a vector to control the expression of the mouse dihydrofolate reductase gene (dhfr). Although all essential parts of this translation initiation region (TIR) should be present, this fragment showed only very low activity. Computer analysis revealed a potentially inhibitory hairpin binding the S - D sequence into its stem base paired to vectorderived upstream sequences. Mutational alterations demonstrated that this hairpin was not responsible for the low activity. However, addition of 21 nt of the T7 gene 1 upstream sequence to the 29 base pair fragment were capable of increasing the translational efficiency by one order of magnitude. Computer analysis of this sequence, including nucleotide snuffling, revealed that it contains a highly unstructured region lacking mRNA secondary structures but with a hairpin at its 5′ end, here formed solely by T7 sequences. There was not much difference in activity whether the mRNA included or lacked vector-derived sequences upstream of the hairpin. Such highly unstructured mRNA regions were found in all very efficiently expressed T7 genes without any obvious sequence homologies. The △G values of these regions were higher, i.e. potential secondary structural elements were fewer, than in TIR of genes from E.coli. This is likely due to the fact that T7 as a lytic phage is relying for successful infection on much stronger signals which a cell cannot afford because of the indispensable balanced equilibria of its interdependent biochemical processes. When the 5′ ends of efficient T7 gene mRNA are formed by the action of RNase III they generally start with an unstructured region. Efficiently expressed T7 genes within a polycistronic mRNA, however, always contain a hairpin preceding the structure free sequence. We suggest that the formation of this 5′ hairpin is releasing enough energy to keep the unstructured regions free of secondary RNA structures for sufficient time to give ribosomes and factors a good chance for binding to the TIR. In addition, sequences further downstream of the start codon give rise to an additional increase in efficiency of the TIR by almost two orders of magnitude.
机译：在该噬菌体的感染周期中，噬菌体T7早期区域的基因1（RNA聚合酶基因）非常活跃地被翻译。将其核糖体结合位点的29个碱基对片段（包含起始三联体，Shine-Dalgarno序列（SD），这些中央元件的上游10个核苷酸（nt）和下游6 n​​t）克隆到载体中，以控制小鼠的表达二氢叶酸还原酶基因（dhfr）。尽管应存在此翻译起始区（TIR）的所有基本部分，但该片段仅显示出非常低的活性。计算机分析揭示了潜在的抑制性发夹，其将S-D序列结合到其茎碱基与载体衍生的上游序列配对。突变表明该发夹与低活性无关。但是，向29个碱基对的片段中添加21 nt的T7基因1上游序列能够将翻译效率提高一个数量级。该序列的计算机分析（包括核苷酸打断）表明，它包含一个高度非结构化的区域，该区域缺少mRNA二级结构，但在5'端带有发夹，此处仅由T7序列形成。无论mRNA是否包含发夹上游的载体衍生序列，活性都没有太大差异。在所有非常有效表达的T7基因中发现了这种高度非结构化的mRNA区，没有任何明显的序列同源性。与来自大肠杆菌的基因的TIR相比，这些区域的△G值更高，即潜在的二级结构元件更少。这可能是由于以下事实：作为裂解性噬菌体的T7依赖于更强的信号成功感染，而这些信号由于其相互依赖的生化过程必不可少的平衡而无法承受。当有效的T7基因mRNA的5'端通过RNase III的作用形成时，它们通常以无结构的区域开始。然而，多顺反子mRNA中有效表达的T7基因始终在结构自由序列之前包含发夹。我们建议，此5'发夹的形成释放足够的能量，以使无结构的区域保持无二级RNA结构的时间足够长，以使核糖体和因子具有与TIR结合的良好机会。另外，在起始密码子的更下游的序列使TIR的效率额外增加了几乎两个数量级。

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