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在大肠杆菌中筛选Tn10四环素阻遏物与四环素操纵子的结合：温度敏感突变体的分离及DNA结合基序中的组合诱变

Selection for Tn10 tet repressor binding to tet operator in Escherichia coli: isolation of temperature-sensitive mutants and combinatorial mutagenesis in the DNA binding motif.

作者信息

Wissmann A, Wray L V, Somaggio U, Baumeister R, Geissendörfer M, Hillen W

机构信息

Lehrstuhl für Mikrobiologie, Friedrich Alexander Universität Erlangen/Nürnberg, Federal Republic of Germany.

出版信息

Genetics. 1991 Jun;128(2):225-32. doi: 10.1093/genetics/128.2.225.

DOI:10.1093/genetics/128.2.225
PMID:1649065
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1204461/
Abstract

We have constructed a genetic assay which selects positively for a functional interaction between Tet repressor and its cognate operator in Escherichia coli. In this strain Tet repressor blocks expression of lacI and lacZ. This leads to derepression of a lacPO controlled galK gene. The strain can be selected by growth on galactose as the sole carbon source and screened for the beta-galactosidase phenotype. These features allow the identification of one candidate among 10(8) false clones on a single plate. The assay was applied to select mutants with a ts DNA binding phenotype and to screen oligonucleotide generated Tet repressor mutants. Analysis of these mutations revealed that they affect DNA and inducer binding and possibly the dimerization domains. These mutations are located at residues 21, 48, 49, 89 and at the C terminus of the protein (193), respectively.

摘要

我们构建了一种遗传检测方法，该方法能在大肠杆菌中对四环素阻遏物与其同源操纵基因之间的功能性相互作用进行正向选择。在这个菌株中，四环素阻遏物会阻断lacI和lacZ的表达。这会导致由lacPO控制的galK基因去阻遏。该菌株可以通过在以半乳糖作为唯一碳源的培养基上生长来进行选择，并针对β - 半乳糖苷酶表型进行筛选。这些特性使得在单个平板上能够从10⁸个假克隆中鉴定出一个候选克隆。该检测方法被用于筛选具有温度敏感型DNA结合表型的突变体以及对寡核苷酸产生的四环素阻遏物突变体进行筛选。对这些突变的分析表明，它们影响DNA和诱导剂结合，并且可能影响二聚化结构域。这些突变分别位于蛋白质的第21、48、49、89位残基以及蛋白质的C末端（193位）。

相似文献

1
Selection for Tn10 tet repressor binding to tet operator in Escherichia coli: isolation of temperature-sensitive mutants and combinatorial mutagenesis in the DNA binding motif.在大肠杆菌中筛选Tn10四环素阻遏物与四环素操纵子的结合：温度敏感突变体的分离及DNA结合基序中的组合诱变
Genetics. 1991 Jun;128(2):225-32. doi: 10.1093/genetics/128.2.225.
2
Quantitative analysis of Tn10 Tet repressor binding to a complete set of tet operator mutants.Tn10 四环素阻遏物与全套四环素操纵子突变体结合的定量分析。
Nucleic Acids Res. 1990 May 25;18(10):2875-80. doi: 10.1093/nar/18.10.2875.
3
Functional roles of amino acid residues involved in forming the alpha-helix-turn-alpha-helix operator DNA binding motif of Tet repressor from Tn10.参与形成来自Tn10的Tet阻遏物的α-螺旋-转角-α-螺旋操纵子DNA结合基序的氨基酸残基的功能作用。
Proteins. 1992 Oct;14(2):168-77. doi: 10.1002/prot.340140204.
4
Contacts between Tet repressor and tet operator revealed by new recognition specificities of single amino acid replacement mutants.通过单个氨基酸替换突变体的新识别特异性揭示 Tet 阻遏物与 tet 操纵子之间的相互作用
J Mol Biol. 1992 Aug 20;226(4):1257-70. doi: 10.1016/0022-2836(92)91065-w.
5
The role of the N terminus in Tet repressor for tet operator binding determined by a mutational analysis.
J Biol Chem. 1992 Jan 25;267(3):1945-52.
6
Saturation mutagenesis of the Tn10-encoded tet operator O1. Identification of base-pairs involved in Tet repressor recognition.
J Mol Biol. 1988 Aug 5;202(3):397-406. doi: 10.1016/0022-2836(88)90273-2.
7
Mutations in the Tn10 tet repressor that interfere with induction. Location of the tetracycline-binding domain.干扰诱导的Tn10四环素阻遏物中的突变。四环素结合结构域的定位。
J Mol Biol. 1988 Oct 20;203(4):949-59. doi: 10.1016/0022-2836(88)90120-9.
8
Noninducible Tet repressor mutations map from the operator binding motif to the C terminus.不可诱导的四环素阻遏物突变从操纵子结合基序映射到C末端。
J Bacteriol. 1993 Feb;175(4):1206-10. doi: 10.1128/jb.175.4.1206-1210.1993.
9
Amino acids determining operator binding specificity in the helix-turn-helix motif of Tn10 Tet repressor.决定Tn10四环素阻遏物螺旋-转角-螺旋基序中操纵子结合特异性的氨基酸。
EMBO J. 1991 Dec;10(13):4145-52. doi: 10.1002/j.1460-2075.1991.tb04992.x.
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Overproduction and purification of the Tn10-specified inner membrane tetracycline resistance protein Tet using fusions to beta-galactosidase.利用与β-半乳糖苷酶的融合蛋白过量生产和纯化Tn10指定的内膜四环素抗性蛋白Tet
Mol Microbiol. 1990 Aug;4(8):1241-51. doi: 10.1111/j.1365-2958.1990.tb00703.x.

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Structural and functional characterization of MrpR, the master repressor of the Bacillus subtilis prophage SPβ.SPβ 噬菌体的主阻遏物 MrpR 的结构和功能特征。
Nucleic Acids Res. 2023 Sep 22;51(17):9452-9474. doi: 10.1093/nar/gkad675.
2
Regulated Expression Systems for Mycobacteria and Their Applications.分枝杆菌的调控表达系统及其应用
Microbiol Spectr. 2014;2(1). doi: 10.1128/microbiolspec.MGM2-0018-2013.
3
A novel TetR-regulating peptide turns off rtTA-mediated activation of gene expression.一种新型的四环素阻遏蛋白（TetR）调控肽可关闭反向四环素转录激活因子（rtTA）介导的基因表达激活。
PLoS One. 2014 May 8;9(5):e96546. doi: 10.1371/journal.pone.0096546. eCollection 2014.
4
Efficient and exclusive induction of Tet repressor by the oligopeptide Tip results from co-variation of their interaction site.寡肽Tip对Tet阻遏物的高效且特异性诱导源于它们相互作用位点的共同变异。
Nucleic Acids Res. 2007;35(12):3945-52. doi: 10.1093/nar/gkm357. Epub 2007 Jun 1.
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Synthetic tetracycline-inducible regulatory networks: computer-aided design of dynamic phenotypes.合成四环素诱导调控网络：动态表型的计算机辅助设计
BMC Syst Biol. 2007 Jan 9;1:7. doi: 10.1186/1752-0509-1-7.
6
Random insertion of a TetR-inducing peptide tag into Escherichia coli proteins allows analysis of protein levels by induction of reporter gene expression.将TetR诱导肽标签随机插入大肠杆菌蛋白中，可通过诱导报告基因表达来分析蛋白水平。
Appl Environ Microbiol. 2006 Aug;72(8):5637-42. doi: 10.1128/AEM.00739-06.
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Independent regulation of two genes in Escherichia coli by tetracyclines and Tet repressor variants.四环素和四环素阻遏物变体对大肠杆菌中两个基因的独立调控
J Bacteriol. 2004 Jul;186(13):4399-401. doi: 10.1128/JB.186.13.4399-4401.2004.
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Two mutations in the tetracycline repressor change the inducer anhydrotetracycline to a corepressor.四环素阻遏物中的两个突变将诱导剂脱水四环素转变为共阻遏物。
Nucleic Acids Res. 2004 Feb 4;32(2):842-7. doi: 10.1093/nar/gkh200. Print 2004.
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Cloning and characterization of SmeT, a repressor of the Stenotrophomonas maltophilia multidrug efflux pump SmeDEF.嗜麦芽窄食单胞菌多药外排泵SmeDEF的阻遏物SmeT的克隆与特性分析
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本文引用的文献

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High-frequency generalised transduction by bacteriophage T4.噬菌体T4介导的高频广义转导
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A perfectly symmetric lac operator binds the lac repressor very tightly.一个完全对称的乳糖操纵子紧密结合乳糖阻遏物。
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Control of expression of the Tn10-encoded tetracycline resistance genes. Equilibrium and kinetic investigation of the regulatory reactions.Tn10编码的四环素抗性基因表达的调控。调控反应的平衡和动力学研究。
J Mol Biol. 1983 Sep 25;169(3):707-21. doi: 10.1016/s0022-2836(83)80166-1.
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Overlapping divergent promoters control expression of Tn10 tetracycline resistance.重叠的反向启动子控制Tn10四环素抗性的表达。
Gene. 1983 Aug;23(2):149-56. doi: 10.1016/0378-1119(83)90046-x.
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pUR 250 allows rapid chemical sequencing of both DNA strands of its inserts.pUR 250可对其插入片段的两条DNA链进行快速化学测序。
Nucleic Acids Res. 1982 Oct 11;10(19):5765-72. doi: 10.1093/nar/10.19.5765.
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A multifunctional gene (tetR) controls Tn10-encoded tetracycline resistance.一个多功能基因（tetR）控制着Tn10编码的四环素抗性。
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