LOCALIZATION AND CHARACTERIZATION OF THE GENE ENCODING RELEASE FACTOR RF3 IN ESCHERICHIA-COLI
LOCALIZATION AND CHARACTERIZATION OF THE GENE ENCODING RELEASE FACTOR RF3 IN ESCHERICHIA-COLI
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DOI：
10.1073/pnas.91.13.5848
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发表时间：
1994-06-21
影响因子：
11.1
通讯作者：
BUCKINGHAM, RH
中科院分区：
文献类型：
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作者：
GRENTZMANN, G;BRECHEMIERBAEY, D;BUCKINGHAM, RH
两个蛋白质释放因子（RFs）显示密码子特异性，RF 1和RF 2，是已知的大肠杆菌多肽链终止所需的。第三种蛋白质组分也被描述为刺激体外终止，但仍然不确定这种蛋白质RF 3是否参与体内终止或对细胞生长至关重要。我们报告了（i）RF 3的纯化和N-末端测序;（ii）分离类似于miaD的转座子插入突变体，miaD是影响基因miaA的泄漏UAA突变的抑制剂，导致增强的无义抑制;（iii）受影响基因在染色体物理图谱上的定位;和（iv）野生型基因的克隆和测序，提供其编码因子RF 3的证据。我们将基因命名为prfC。两个转座子插入显示在密码子287和426处中断prfC的编码序列。插入突变体中增强的无义抑制表明该产物参与体内终止。这种突变体的分离强烈表明，基因产物对细胞活力不是必需的，尽管细胞生长受到影响。RF 3是一种分子量为59，460的蛋白质，含有528个氨基酸，与延伸因子EF-G（核糖体转位所必需的GTP结合蛋白）和其他已知或认为与核糖体相互作用的GTP结合蛋白非常相似。
Two protein release factors (RFs) showing codon specificity, RF1 and RF2, are known to be required for polypeptide chain termination in Escherichia coli. A third protein component has also been described that stimulates termination in vitro, but it has remained uncertain whether this protein, RF3, participates in termination in vivo or is essential to cell growth. We report (i) the purification and N-terminal sequencing of RF3; (ii) the isolation of transposon insertion mutants similar to miaD, a suppressor of a leaky UAA mutation affecting the gene miaA, leading to enhanced nonsense suppression; (iii) the localization of the affected gene on the physical map of the chromosome; and (iv) the cloning and sequencing of the wild-type gene, providing proof that it encodes the factor RF3. We designate the gene prfC. Two transposon insertions were shown to interrupt the coding sequence of prfC, at codons 287 and 426. The enhanced nonsense suppression in the insertion mutants shows that the product participates in termination in vivo. The isolation of such mutants strongly suggests that the gene product is not essential to cell viability, though cell growth is affected. RF3 is a protein with a molecular weight of 59,460 containing 528 amino acids and displays much similarity to elongation factor EF-G, a GTP binding protein necessary for ribosomal translocation, and other GTP binding proteins known or thought to interact with the ribosome.