摘要

       含大肠杆菌精氨酰tRNA合成酶 (ArgRS)基因 (argS)的 pUC18重组质粒 ,在大肠杆菌TG1转化子中能够高表达ArgRS近 10 0 0倍。为了研究大肠杆菌argS的表达调控 ,构建了一系列的缺失突变。分别缺失全部上游序列 (argSΔ1)、Shine Dalgarno(SD)区 (argSΔ2 )和缺失启动子 - 10区下游 (相当于翻译起始位点 - 6 5nt ,argSΔ3)前的上游序列后的变种argS都不能表达ArgRS。而缺失 - 12 2nt (距翻译起始位点 - 180nt,argSΔ8)、- 70nt (距翻译起始位点 - 12 8nt,argSΔ7)、- 5 2nt (距翻译起始位点 - 110nt ,argSΔ6 )、- 35区 (距翻译起始位点 - 94nt,argSΔ5 )、启动子 - 10区 (距翻译起始位点 - 71nt,argSΔ4)前的上游序列后 ,这些缺失突变基因的表达水平与野生型argS接近。但argSΔ4、argSΔ5、argSΔ6都会形成部分包涵体。通过RNA斑点杂交测定发现 ,argSΔ4、argSΔ5和argSΔ6的mRNA转录量为argS及argSΔ7的 2到3倍。即 - 5 2nt和 - 70nt之间的 19个碱基 (AATAGTGAAAACGGCAATA)可能是大肠杆菌argS的转录负调控区。该元件的缺失将使得ArgRS过快表达并导致部分蛋白质形成包涵体。凝胶阻滞分析也发现在细胞粗抽液中有一个因子可以专一地与这个负调控元件结合 ,它可能参与该基因的表达调控。精氨酸专一性地诱导argS

       The pUC18 recombinant plasmid containing the Escherichia coli arginyl tRNA synthetase (ArgRS) gene (argS) can express nearly 100 times ArgRS in Escherichia coli TG1 transformants. In order to study the expression regulation of E. coli argS, a series of deletion mutations were constructed. The variant argS after deletion of all upstream sequences (argSΔ1), Shine Dalgarno (SD) region (argSΔ2) and the upstream sequence before the deletion of the promoter-10 region downstream (equivalent to the translation start site-6 5nt, argSΔ3), respectively Express ArgRS. And the deletion-12 2nt (from the translation start site-180nt, argSΔ8),-70nt (from the translation start site-12 8nt, argSΔ7),-5 2nt (from the translation start site-110nt, argSΔ6), -35 region (from the translation initiation site-94nt, argSΔ5), the promoter-10 region (from the translation initiation site-71nt, argSΔ4) after the upstream sequence, the expression level of these deletion mutant genes and wild-type argS near. However, argSΔ4, argSΔ5, and argSΔ6 all formed partial inclusion bodies. The RNA dot blot assay showed that the mRNA transcripts of argSΔ4, argSΔ5 and argSΔ6 were 2 to 3 times that of argS and argSΔ7. That is, the 19 bases between -52nt and -70nt (AATAGTGAAAACGGCAATA) may be the negative transcriptional regulatory region of E. coli argS. The deletion of this element will cause ArgRS to express too quickly and cause some proteins to form inclusion bodies. Gel block analysis also found that there is a factor in the crude cell aspirate that can specifically bind to this negative regulatory element, and it may participate in the regulation of the gene expression. Arginine specifically induces argS

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