...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Nucleic Acids Research >IN VITRO AND IN VIVO FUNCTION OF THE C-TERMINUS OF ESCHERICHIA COLI SINGLE-STRANDED DNA BINDING PROTEIN
【24h】

IN VITRO AND IN VIVO FUNCTION OF THE C-TERMINUS OF ESCHERICHIA COLI SINGLE-STRANDED DNA BINDING PROTEIN

机译：大肠埃希菌单链DNA结合蛋白C末端的体外和体内功能

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

We constructed several deletion mutants of Escherichia coli single-stranded DNA binding protein (EcoSSB) lacking different parts of the C-terminal region. This region of EcoSSB is composed of two parts: a glycine- and proline-rich sequence of -60 amino acids followed by an acidic region of the last 10 amino acids which is highly conserved among the bacterial SSB proteins. The single-stranded DNA binding protein of human mitochondria (HsmtSSB) lacks a region homologous to the C-terminal third of EcoSSB. Therefore, we also investigated a chimeric protein consisting of the complete sequence of the human mitochondrial single-stranded DNA binding protein (HsmtSSB) and the C-terminal third of EcoSSB. Fluorescence titrations and DNA-melting curves showed that the C-terminal third of EcoSSB is not essential for DNA-binding in vitro. The affinity for single-stranded DNA and RNA is even increased by the removal of the last 10 amino acids. Consequently, the nucleic acid binding affinity of HsmtSSB is reduced by the addition of the C-terminus of EcoSSB. All mutant proteins lacking the last 10 amino acids are unable to substitute wild-type EcoSSB in vivo. Thus, while the nucleic acid binding properties do not depend on an intact C-terminus, this region is essential for in vivo function. Although the BMA binding properties of HsmtSSB and EcoSSB are quite similar, HsmtSSB does not function in E.coli. This failure cannot be overcome by fusing the C-terminal third of EcoSSB to HsmtSSB. Thus differences in the N-terminal parts of both proteins must be responsible for this incompatibility. None of the mutants was defective in tetramerization. However, mixed tetramers could only be formed by proteins containing the same N-terminal part. This reflects structural differences between the N-terminal parts of HsmtSSB and EcoSSB. These results indicate that the region of the last 10 amine acids, which is highly conserved among bacterial SSB proteins, is involved in essential protein-protein interactions in the E.coli cell.
机译：我们构建了几个缺失突变体的大肠杆菌单链DNA结合蛋白（EcoSSB），缺少C端区域的不同部分。 EcoSSB的这一区域由两部分组成：-60个氨基酸的富含甘氨酸和脯氨酸的序列，其后是在细菌SSB蛋白中高度保守的最后10个氨基酸的酸性区域。人线粒体（HsmtSSB）的单链DNA结合蛋白缺少与EcoSSB的C末端三分之一同源的区域。因此，我们还研究了由人线粒体单链DNA结合蛋白（HsmtSSB）的完整序列和EcoSSB的C端三分之一组成的嵌合蛋白。荧光滴定和DNA熔解曲线表明，EcoSSB的C末端三分之一对于体外DNA结合不是必需的。通过去除最后10个氨基酸，甚至增加了对单链DNA和RNA的亲和力。因此，通过添加EcoSSB的C末端降低了HsmtSSB的核酸结合亲和力。缺少最后10个氨基酸的所有突变蛋白都无法在体内替代野生型EcoSSB。因此，尽管核酸结合特性不取决于完整的C末端，但是该区域对于体内功能是必不可少的。尽管HsmtSSB和EcoSSB的BMA绑定属性非常相似，但是HsmtSSB在大肠杆菌中不起作用。通过将EcoSSB的C端三分之一融合到HsmtSSB不能克服此故障。因此，两种蛋白质N末端部分的差异必须是造成这种不相容性的原因。所有突变体均无四聚体缺陷。但是，混合四聚体只能由包含相同N末端部分的蛋白质形成。这反映了HsmtSSB和EcoSSB的N端部分之间的结构差异。这些结果表明，在细菌SSB蛋白之间高度保守的最后10个胺酸的区域参与大肠杆菌细胞中必需的蛋白-蛋白相互作用。

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