Identification of mammalian mitochondrial translational initiation factor 3 and examination of its role in initiation complex formation with natural mRNAs
Identification of mammalian mitochondrial translational initiation factor 3 and examination of its role in initiation complex formation with natural mRNAs
复制标题
DOI：
10.1074/jbc.m202498200
复制
发表时间：
2002-09-20
影响因子：
4.8
通讯作者：
Spremulli, LL
中科院分区：
文献类型：
--
作者：
Koc, EC;Spremulli, LL
人线粒体翻译起始因子3（IF 3（mt））已从人表达序列标签数据库中鉴定。使用来自细菌IF 3保守区域的共有序列，鉴定了几个部分测序的cDNA克隆，并从重叠克隆中通过计算机组装了完整序列。IF 3（mt）全长278个氨基酸残基。MitoProt II预测该蛋白质将定位于线粒体中的概率为97%，并进一步预测成熟蛋白质的长度为247个残基。克隆预测的IF 3（mt）成熟形式的cDNA，并在大肠杆菌中以His标记形式表达蛋白质。IF 3的成熟形式（mt）在与细菌IF 3同源的区域周围的N和C末端上具有短的延伸。与原核因子同源的IF 3（mt）区域与细菌蛋白质的同源性在21-26%之间。在存在线粒体起始因子2（IF 2（mt））、[S-35]fMet-tRNA和poly（A，U，G）或细胞色素氧化酶亚基II基因的体外转录物（mRNA）的情况下，纯化IF 3（mt）可促进线粒体55 S核糖体上起始复合物的形成。IF 3（mt）使55 S线粒体核糖体与其亚基之间的平衡向亚基解离方向移动。此外，E. coli起始因子1刺激E.在IF 2（mt）和IF 3（mt）存在下，研究了大肠杆菌70 S和线粒体55 S核糖体的相互作用。
Human mitochondrial translational initiation factor 3 (IF3(mt)) has been identified from the human expressed sequence tag data base. Using consensus sequences derived from conserved regions of the bacterial IF3, several partially sequenced cDNA clones were identified, and the complete sequence was assembled in silico from overlapping clones. IF3(mt) is 278 amino acid residues in length. MitoProt II predicts a 97% probability that this protein will be localized in mitochondria and further predicts that the mature protein will be 247 residues in length. The cDNA for the predicted mature form of IF3(mt) was cloned, and the protein was expressed in Escherichia coli in a His-tagged form. The mature form of IF3(mt) has short extensions on the N and C termini surrounding a region homologous to bacterial IF3. The region of IF3(mt) homologous to prokaryotic factors ranges between 21-26% identical to the bacterial proteins. Purified IF3(mt) promotes initiation complex formation on mitochondrial 55 S ribosomes in the presence of mitochondrial initiation factor 2 (IF2(mt)), [S-35]fMet-tRNA, and either poly(A,U,G) or an in vitro transcript of the cytochrome oxidase subunit II gene as mRNA. IF3(mt) shifts the equilibrium between the 55 S mitochondrial ribosome and its subunits toward subunit dissociation. In addition, the ability of E. coli initiation factor 1 to stimulate initiation complex formation on E. coli 70 S and mitochondrial 55 S ribosomes was investigated in the presence of IF2(mt) and IF3(mt).