Mutations in the highly conserved GGQ motif of class 1 polypeptide release factors abolish ability of human eRF1 to trigger peptidyl-tRNA hydrolysis
Mutations in the highly conserved GGQ motif of class 1 polypeptide release factors abolish ability of human eRF1 to trigger peptidyl-tRNA hydrolysis
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DOI：
10.1017/s135583829999043x
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发表时间：
1999-08-01
期刊：
影响因子：
4.5
通讯作者：
Kisselev, LL
中科院分区：
文献类型：
--
作者：
Frolova, LY;Tsivkovskii, RY;Kisselev, LL
虽然1类多肽释放因子（原核生物中的681和RF 2，真核生物中的eRF 1）的一级结构是已知的，但它们在翻译终止中起作用的分子基础仍然不清楚。因为所有1类RF都促进肽基-tRNA的终止密码子依赖性和核糖体依赖性水解，所以可以预期这种共同功能依赖于共同的结构基序。我们已经比较了可用的1类RF的氨基酸序列，并发现了一个新的，共同的，独特的，严格保守的Coo基序，应该是在一个环（线圈）的构象推断的程序预测蛋白质二级结构。作为1类RF代表的人eRF 1的定点诱变显示，该基序中的两个甘氨酰残基G183和G184的取代导致蛋白质作为释放因子朝向所有三个终止密码子完全失活，而两个相邻的氨基酸残基G181和R182在功能上是非必需的。无活性的人eRF 1突变体在释放测定中与野生型eRF 1竞争，并强烈抑制其释放活性。该基序中甘氨酰残基的突变不影响另一种功能，即eRF 1与核糖体一起诱导人eRF 3（一种2类RF）的GT3活性的能力。我们假设，新的高度保守的GGQ基序直接或间接地牵连在翻译终止的1类RF的活动。
Although the primary structures of class 1 polypeptide release factors (681 and RF2 in prokaryotes, eRF1 in eukaryotes) are known, the molecular basis by which they function in translational termination remains obscure. Because all class 1 RFs promote a stop-codon-dependent and ribosome-dependent hydrolysis of peptidyl-tRNAs, one may anticipate that this common function relies on a common structural motif(s). We have compared amino acid sequences of the available class 1 RFs and found a novel, common, unique, and strictly conserved Coo motif that should be in a loop (coil) conformation as deduced by programs predicting protein secondary structure. Site-directed mutagenesis of the human eRF1 as a representative of class 1 RFs shows that substitution of both glycyl residues in this motif, G183 and G184, causes complete inactivation of the protein as a release factor toward all three stop codons, whereas two adjacent amino acid residues, G181 and R182, are functionally nonessential. Inactive human eRF1 mutants compete in release assays with wild-type eRF1 and strongly inhibit their release activity. Mutations of the glycyl residues in this motif do not affect another function, the ability of eRF1 together with the ribosome to induce GTPase activity of human eRF3, a class 2 RF. We assume that the novel highly conserved GGQ motif is implicated directly or indirectly in the activity of class 1 RFs in translation termination.