TRANSCRIPTION ATTENUATION-MEDIATED CONTROL OF LEU OPERON EXPRESSION - INFLUENCE OF THE NUMBER OF LEU CONTROL CODONS
TRANSCRIPTION ATTENUATION-MEDIATED CONTROL OF LEU OPERON EXPRESSION - INFLUENCE OF THE NUMBER OF LEU CONTROL CODONS
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DOI：
10.1128/jb.173.5.1634-1641.1991
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发表时间：
1991-03-01
影响因子：
3.2
通讯作者：
CALVO, JM
中科院分区：
文献类型：
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作者：
BARTKUS, JM;TYLER, B;CALVO, JM
通过定点诱变改变鼠伤寒沙门氏菌的前导区，以产生具有 1 至 7 个相邻 Leu 密码子（全部为 CUA）的构建体。在含有六个这样的构建体的菌株中测量了 leu 操纵子的表达，每个构建体以单个拷贝的形式整合到染色体中。操纵子表达足够高，使得所有菌株都在未补充亮氨酸的基本培养基中生长。在以这样的方式培养的菌株中测量操纵子的表达，即它们的生长受到细胞内亮氨酸或亮氨酰-tRNA浓度的限制。一般来说，每个构建体的亮氨酸操纵子在作为限制程度的函数的表达程度方面与亲本构建体的反应相似。然而，含有(CUA)1的菌株以及在一定程度上含有(CUA)2的菌株的反应稍微迟缓，而含有(CUA)6和(CUA)7的菌株对限制的反应比亲本构建体更敏感。此外，含有 leu 启动子和前导区的 DNA 片段在使用纯化的 RNA 聚合酶的体外转录反应中用作模板。当三磷酸核苷浓度为 200-mu-M 时，RNA 聚合酶在 leu 前导区转录过程中在转录起始位点下游约 95 bp 的位点处暂停。暂停的中场时间在 37 摄氏度时为 1 分钟，在 22 摄氏度时为 3 分钟。当 GTP 浓度降低至 20-mu-M 时，暂停时间显着延长。我们的结果最容易用全有或全无模型来解释。给定两个亮氨酸控制密码子，操纵子在大范围的亮氨酸限制下以几乎最大输出做出响应，并且该结果不会随着控制密码子数量的增加而改变太大。
The leader region from S. typhimurium was altered by site-directed mutagenesis to produce constructs having between one and seven adjacent Leu codons, all CUA. leu operon expression was measured in strains containing six of these constructs, each integrated into the chromosome in a single copy. Operon expression was sufficiently high that all strains grew in minimal medium unsupplemented by leucine. Expression of the operon was measured in strains cultured in such a way that their growth was limited by the intracellular concentration of either leucine or of leucyl-tRNA. In general, the leu operon for each construct responded similarly to the parent construct in terms of the degree of expression as a function of the degree of limitation. However, a strain containing (CUA)1 and, to a certain extent, a strain having (CUA)2 responded somewhat more sluggishly and strains containing (CUA)6 and (CUA)7 responded more sensitively to limitations than did the parent construct. In addition, DNA fragments containing the leu promoter and leader region were used as templates in in vitro transcription reactions employing purified RNA polymerase. With nucleoside triphosphate concentrations of 200-mu-M, RNA polymerase paused during transcription of the leu leader region at a site about 95 bp downstream from the site of transcription initiation. The halftimes of the pause were 1 min at 37-degrees-C and 3 min at 22-degrees-C. The pause was lengthened substantially when the GTP concentration was lowered to 20-mu-M. Our results are interpreted most easily in terms of an all-or-none model. Given two Leu control codons, the operon responds with nearly maximum output over a wide range of leucine limitation, and that outcome does not change much with increasing numbers of control codons.