Functional characterization of yeast mitochondrial release factor 1
Functional characterization of yeast mitochondrial release factor 1
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DOI：
10.1074/jbc.m910448199
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发表时间：
2000-06-09
影响因子：
4.8
通讯作者：
Tate, WP
中科院分区：
文献类型：
--
作者：
Askarian-Amiri, ME;Pel, HJ;Tate, WP
从克隆的MRF1基因中表达了酿酒酵母线粒体释放因子，并从包涵体中纯化，复性后具有功能活性。该基因编码一种具有释放活性的因子，在与线粒体核糖体的终止试验和与大肠杆菌核糖体的试验中识别同源终止密码子。在异源分析中，线粒体中编码色氨酸的非同源终止密码子UGA被微弱识别。线粒体释放因子1蛋白与细菌核糖体结合，并与终止复合体中结合的信使核糖核酸中的终止密码子形成交联。亲和力强烈依赖于停止信号的同一性。克隆了MRF1的两个等位基因，一个是一级结构释放因子1的点突变，另一个是在体内补偿线粒体核糖体rRNA解码点突变的等位基因，并对表达的蛋白质进行纯化和复性，与线粒体释放因子1相比，差异蛋白与核糖体的结合受损，释放因子中的这个结构区域可能参与了密码子依赖性的特异性核糖体相互作用。
The yeast Saccharomyces cerevisiae mitochondrial release factor was expressed from the cloned MRF1 gene, purified from inclusion bodies, and refolded to give functional activity. The gene encoded a factor with release activity that recognized cognate stop codons in a termination assay with mitochondrial ribosomes and in an assay with Escherichia coli ribosomes. The noncognate stop codon, UGA, encoding tryptophan in mitochondria, was recognized weakly in the heterologous assay. The mitochondrial release factor 1 protein bound to bacterial ribosomes and formed a cross-link with the stop codon within a mRNA bound in a termination complex. The affinity was strongly dependent on the identity of stop signal. Two alleles of MRF1 that contained point mutations in a release factor 1 specific region of the primary structure and that in vivo compensated for mutations in the decoding site rRNA of mitochondrial ribosomes were cloned, and the expressed proteins were purified and refolded, The variant proteins showed impaired binding to the ribosome compared with mitochondrial release factor 1, This structural region in release factors is likely to be involved in codon-dependent specific ribosomal interactions.