Escherichia coli RNA polymerase binding sites and transcription initiation sites in the transposon Tn3.
Escherichia coli RNA polymerase binding sites and transcription initiation sites in the transposon Tn3.
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大肠杆菌 RNA 聚合酶结合位点和转座子 Tn3 中的转录起始位点。
DOI：
10.1016/0378-1119(83)90135-x
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发表时间：
1983
期刊：
影响因子：
3.5
通讯作者：
Ohtsubo,E
中科院分区：
文献类型：
--
作者：
Wishart,WL;Machida,C;Ohtsubo,H;Ohtsubo,E
我们已经确定了转座子Tn 3中的大肠杆菌RNA聚合酶结合位点和转录起始位点。硝酸纤维素滤膜结合试验的结果表明，有两个区域内的Tn 3能够形成稳定的二元复合物与RNA聚合酶。这两个区域是含有转座酶（tnpA）和阻遏物（tnpR）基因的N-末端编码序列的208-bp区域，以及含有β-内酰胺酶（bla）基因的N-末端编码序列的332-bp区域。208-bp和332-bp片段的DNA酶I足迹分析进一步确定了延伸的保护区域，约为100 bp。110 bp长，位于转座酶和阻遏物编码区之间，以及β-内酰胺酶基因N-末端编码序列附近的80 bp保护区。在体外转录研究的片段含有这些保护区，使我们能够确定转座酶，阻遏蛋白和β-内酰胺酶mRNA的精确转录起始位点。转座酶和阻遏物mRNA转录发散，它们的转录起始位点相隔80 bp。转座酶和阻遏物启动子的-35同源区相隔10 bp，转座酶启动子的-10同源区与阻遏物蛋白的位点特异性重组酶活性（解离酶）的重组位点（res）一致，这是Tn 3共整合体的解析所必需的。我们讨论了这个复杂的分歧转录的启动子区域的意义，就调节Tn 3转座，我们提出了一个协调调节thetnpAandtnpRgenes的模型。我们还比较了Tn 3 tnpA-tnpR顺反子间区与密切相关的转座子γδ的顺反子间区。
We have identified theEscherichia coliRNA polymerase-binding sites and the transcription initiation sites in the transposon Tn3. Results from nitrocellulose filter-binding assays indicate that there are two regions within Tn 3 capable of forming stable binary complexes with RNA polymerase. The two regions are a 208-bp region containing the N-terminal coding sequence of the transposase (tnpA) and repressor (tnpR) genes, and a 332-bp region containing the N-terminal coding sequence for the β-lactamase (bla) gene. DNase I footprint analysis of the 208-bp and 332-bp fragments further defined an extended region of protection, approx. 110 bp long, located between the transposase and repressor coding regions, and an 80-bp region of protection near the N-terminal coding sequence of the β-lactamase gene. In vitro transcription studies with fragments containing these protected regions allowed us to determine the precise transcription initiation sites for the transposase, repressor, and β-lactamase mRNAs. The transposase and repressor mRNAs are transcribed divergently and their transcription initiation sites are separated by 80 bp. The −35 homology regions for the transposase and repressor promoters are separated by 10 bp and the −10 homology region of the transposase promoter is coincident with the recombination site (res) for the site-specific recombinase activity (resolvase) of the repressor protein, which is required for resolution of Tn 3 cointegrates. We discuss the significance of this complex divergently transcribed promoter region with respect to regulation of Tn3 transposition and we propose a model for coordinated regulation of thetnpAandtnpRgenes. We also compare the Tn3tnpA-tnpRintercistronic region with that of the closely related transposon γδ.