Analysis of the interaction of 16S rRNA and cytoplasmic membrane with the C-terminal part of the Streptococcus pneumoniae Era GTPase
Analysis of the interaction of 16S rRNA and cytoplasmic membrane with the C-terminal part of the Streptococcus pneumoniae Era GTPase
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DOI：
10.1046/j.1432-1033.2001.02493.x
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发表时间：
2001-11-01
期刊：
影响因子：
--
通讯作者：
Zhao, G
中科院分区：
文献类型：
--
作者：
Hang, JQ;Meier, TI;Zhao, G
Era是一种重要的GTPase，在几个细胞过程中起调节作用。肺炎链球菌的Era蛋白最近被证明与16S rRNA和细胞质膜结合。然而，负责RNA和膜结合的Era的确切位置尚不清楚。为了确定Era中与RNA和膜相互作用的区域，我们系统地删除了肺炎链球菌Era的c端部分，而保留了负责蛋白质GTPase活性的n端部分。对截断的Era蛋白进行了纯化和表征。c端缺失9或19个氨基酸残基不影响16S rrna的结合活性，而c端进一步缺失（29-114个氨基酸残基）则消除了16S rrna的结合活性。这些结果表明；假设的Era的KH结构域的完整性，横跨c端大约22-83个氨基酸残基，是16S rrna结合所必需的。此外，从c端缺失多达45个残基的Era蛋白保留了膜结合活性，但较长的缺失显著降低了活性。这些结果表明，部分假定的KH结构域也是膜结合所必需的。因此，这些结果首次表明，Era的膜和16S rrna结合活性的关键区域重叠。从其3'端缺失9或19个密码子的era基因补充了缺乏era的大肠杆菌突变株，而较长缺失的基因则没有这样做，从而表明KH结构域对era功能至关重要。综上所述，本研究的结果表明，假定的KH结构域是16S rrna结合活性所必需的，而部分KH结构域也是膜结合活性所必需的。结果还表明Era和16S rRNA之间的相互作用对细菌生长至关重要。
Era, an essential GTPase, plays a regulatory role in several cellular processes. The Era protein of Streptococcus pneumoniae has recently been shown to, bind to 16S rRNA and the cytoplasmic membrane. However, exact locations of Era responsible for RNA- and membrane-binding were unknown. To identify the regions in Era that interact with the RNA and membrane, the C-terminal part of S. pneumoniae Era was systematically deleted while the N-terminal part, responsible for the GTPase activity of the protein, was kept intact. The resulting truncated Era proteins were purified and characterized. The C-terminal deletion of 9 or 19 amino-acid residues did not affect 16S rRNA-binding activity while further deletions of the C-terminus (29-114 amino-acid residues) abolished the activity. These results indicate; that the integrity of the putative KH domain of Era, spanning the amino-acid residues between approximate to 22-83 from the C-terminus, is required for 16S rRNA-binding. Furthermore, the Era proteins with a deletion up to 45 residues from the C-terminus retained membrane-binding activity, but longer deletions significantly reduced the activity. These results indicate that part of the putative KH domain is also required for membrane-binding. Thus, these results indicate for the first time that the regions critical for the membrane- and 16S rRNA-binding activities of Era overlap. The era gene with a deletion of 9 or 19 codons from its 3' terminus complemented an Escherishia coli mutant strain deficient in Era production whereas the genes with longer deletions failed to do so, thereby indicating that the KH domain is essential for Era function. Taken together, the results of this study indicate that the putative KH domain is required for 16S rRNA-binding activity and that part of the KH domain is also required for membrane-binding activity. The results also suggest that the interaction between Era and 16S rRNA is essential for bacterial growth.