• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Plasmid: An International Journal Devoted to Extrachromosomal Gene Systems >EXPRESSION IN ESCHERICHIA COLI OF Y5-MUTANT AND N-TERMINAL DOMAIN-DELETED DNA GYRASE B PROTEINS AFFECTS STRONGLY PLASMID MAINTENANCE
【24h】

EXPRESSION IN ESCHERICHIA COLI OF Y5-MUTANT AND N-TERMINAL DOMAIN-DELETED DNA GYRASE B PROTEINS AFFECTS STRONGLY PLASMID MAINTENANCE

机译：Y5突变株和N端域缺失的DNA旋回酶B蛋白在大肠杆菌中的表达强烈影响质粒的维持。

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Escherichia coli DNA gyrase B subunit (GyrB) is composed of a 43-kDa. N-terminal domain containing an ATP-binding site and a 47-kDa C-terminal domain involved in the interaction with the gyrase A subunit (GyrA). Site-directed mutagenesis was used to substitute, in both the entire GyrB subunit and its 43-kDa N-terminal fragment, the amino acid Y5 by either a serine (Y5S) or a phenylalanine residue (Y5F). Under standard conditions, cells bearing Y5S or Y5F mutant GyrB expression plasmids produced significantly less recombinant proteins than cells transformed with the wild-type plasmid. This dramatic decrease in expression of mutant GyrB proteins was not observed when the corresponding N-terminal 43-kDa mutant plasmids were used. Examination of the plasmid content of the transformed cells after induction showed that the Y5F and Y5S GyrB protein level was correlated with the plasmid copy number. By repressing tightly the promoter activity encoded by these expression vectors during cell growth, it was possible to restore the normal level of the mutant GyrB encoding plasmids in the transformed bacteria. Treatment with chloramphenicol before protein induction enabled large overexpression of the GyrB mutant Y5F and Y5S proteins. Ln addition, the decrease in plasmid copy number was also observed when the 47-kDa C-terminal fragment of the GyrB subunit was expressed in bacteria grown under standard culture conditions. Analysis of DNA supercoiling and relaxation activities in the presence of GyrA demonstrated that purified Y5-mutant GyrB proteins were deficient for ATP-dependent gyrase activities. Taken together, these results show that Y5F and Y5S mutant GyrB proteins, but not the corresponding 43-kDa N-terminal fragments, compete in vivo with the bacterial endogenous GyrB subunit of DNA gyrase, thereby reducing the plasmid copy number in the transformed bacteria by probably acting on the level of negative DNA supercoiling in vivo. This competition could be mediated by the presence of the intact 47-kDa C-terminal domain in the Y5F and Y5S mutant GyrB subunits. This study demonstrates also that the amino acid Y5 is a crucial residue for the expression of the gyrase B activity in vivo. Thus, our in vivo approach may also be useful for detecting other important amino acids for DNA gyrase activity, as mutations affecting the ATPase activity or the GyrB/GyrB or GyrB/GyrA protein interactions. (C) 1997 Academic Press. [References: 38]
机译：大肠杆菌DNA促旋酶B亚基（GyrB）由43 kDa组成。 N末端结构域，包含一个ATP结合位点和一个47 kDa C末端结构域，参与与回旋酶A亚基（GyrA）的相互作用。定点诱变用于在整个GyrB亚基及其43 kDa N端片段中用丝氨酸（Y5S）或苯丙氨酸残基（Y5F）取代氨基酸Y5。在标准条件下，带有Y5S或Y5F突变GyrB表达质粒的细胞产生的重组蛋白比野生型质粒转化的细胞少得多。当使用相应的N端43-kDa突变质粒时，未观察到突变GyrB蛋白表达的急剧下降。诱导后检查转化细胞的质粒含量表明Y5F和Y5S GyrB蛋白水平与质粒拷贝数相关。通过在细胞生长期间紧密抑制这些表达载体编码的启动子活性，有可能恢复转化细菌中突变的GyrB编码质粒的正常水平。在蛋白诱导之前用氯霉素处理使得GyrB突变体Y5F和Y5S蛋白大量过量表达。另外，当在标准培养条件下生长的细菌中表达GyrB亚基的47-kDa C-末端片段时，也观察到质粒拷贝数的减少。在GyrA存在下对DNA超螺旋和弛豫活性的分析表明，纯化的Y5突变型GyrB蛋白缺乏ATP依赖的促旋酶活性。综上所述，这些结果表明Y5F和Y5S突变体GyrB蛋白在体内与DNA促旋酶的细菌内源性GyrB亚基竞争，而不是相应的43-kDa N-末端片段，从而通过转化的细菌减少了质粒拷贝数。可能作用于体内负DNA超螺旋的水平。 Y5F和Y5S突变GyrB亚基中完整的47 kDa C末端结构域的存在可以介导这种竞争。该研究还证明了氨基酸Y5是体内表达促旋酶B活性的关键残基。因此，我们的体内方法也可能用于检测DNA促旋酶活性的其他重要氨基酸，因为这些突变会影响ATPase活性或GyrB / GyrB或GyrB / GyrA蛋白相互作用。 （C）1997学术出版社。 [参考：38]

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号