Delineation of two distinct regulatory domains in the 5' region of the nar operon of Escherichia coli.
Delineation of two distinct regulatory domains in the 5' region of the nar operon of Escherichia coli.
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大肠杆菌 nar 操纵子 5 区域中两个不同调节域的描绘。
DOI：
10.1128/jb.164.1.25-32.1985
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发表时间：
1985
影响因子：
3.2
通讯作者：
DeMoss,JA
中科院分区：
文献类型：
--
作者：
Li,S;Rabi,T;DeMoss,JA
确定了包含大肠杆菌 nar 操纵子 5' 调节区和启动子区的 8.4 KB DNA 片段的详细限制性位点图谱。 nar操纵子的5'末端被亚克隆为2.5-kilobase片段，并且由该亚克隆片段和包含nar操纵子的剩余部分的EcoRI片段构建完整的nar操纵子。选择、定位和表征了一组延伸至完整操纵子 5' 区域的 Bal 31 缺失。基于 nar::Tn5 突变体中硝酸还原酶的 α 和 β 亚基的合成，发现了三类缺失：(i) 允许正常表达的缺失，(ii) 完全阻止表达的缺失，以及 (iii) 允许操纵子厌氧表达但阻止硝酸盐任何额外诱导的缺失。确定nar启动子区的一段核苷酸序列，该片段从后一个删除终点之一开始延伸到操纵子的第一个结构基因中。通过鉴定硝酸还原酶α亚基的前20个N端氨基酸残基的核苷酸序列来定义相对于第一个结构基因narG的翻译起始位点的删除终点的位置。 narG 翻译起始位点上游 161 个碱基对 (bp) 和约 200 bp 处的缺失允许硝酸还原酶的厌氧形成，但干扰硝酸盐对 nar 操纵子表达的刺激。调节区的最大大小由两个 Tn5 插入定义，其位于翻译起始位点 5' 处约 550 bp，并且在有或没有硝酸盐的厌氧条件下不会干扰硝酸盐还原酶的正常表达。我们得出结论，nar操纵子5'调控区分为两个不同的区域：紧邻narG基因5'的100至150 bp包括转录起始位点和操纵子厌氧表达所需的信号，而硝酸盐刺激操纵子表达需要50至400 bp的相邻区域。
A detailed restriction site map was determined for an 8.4-kilobase DNA fragment containing the 5' regulatory and promoter region of the nar operon of Escherichia coli. The 5' end of the nar operon was subcloned as a 2.5-kilobase fragment, and an intact nar operon was constructed from this subcloned fragment and an EcoRI fragment containing the remainder of the nar operon. A set of Bal 31 deletions extending into the 5' region of the intact operon was selected, mapped, and characterized. Based on the synthesis of the alpha and beta subunits of nitrate reductase in a nar::Tn5 mutant, three categories of deletions were found: (i) those which permitted normal expression, (ii) those which completely prevented expression, and (iii) those which permitted anaerobic expression of the operon but prevented any additional induction by nitrate. The nucleotide sequence was determined for a segment of the nar promoter region starting at one of the latter deletion end points and extending into the first structural gene of the operon. The position of the deletion end point relative to the translation start site for the first structural gene, narG, was defined by identifying the nucleotide sequence for the first 20 N-terminal amino acid residues of the alpha subunit of nitrate reductase. Deletions terminating 161 base pairs (bp) and approximately 200 bp upstream from the narG translation start site permitted anaerobic formation of nitrate reductase but interfered with the stimulation of nar operon expression by nitrate. A maximum size for the regulatory region was defined by two Tn5 insertions, which mapped approximately 550 bp 5' from the translation start site and did not interfere with the normal expression of nitrate reductase under anaerobic conditions with or without nitrate. We conclude that the nar operon 5' regulatory region is divided into two distinct regions: the 100 to 150 bp immediately 5' to the narG gene include a transcriptional start site and the signals necessary for anaerobic expression of the operon, and an adjacent region of 50 to 400 bp is required for the stimulation of operon expression by nitrate.