Fan-Minogue Hua, Du Ming, Pisarev Andrey V, Kallmeyer Adam K, Salas-Marco Joe, Keeling Kim M, Thompson Sunnie R, Pestova Tatyana V, Bedwell David M
Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Mol Cell. 2008 Jun 6;30(5):599-609. doi: 10.1016/j.molcel.2008.03.020.
Organisms that use the standard genetic code recognize UAA, UAG, and UGA as stop codons, whereas variant code species frequently alter this pattern of stop codon recognition. We previously demonstrated that a hybrid eRF1 carrying the Euplotes octocarinatus domain 1 fused to Saccharomyces cerevisiae domains 2 and 3 (Eo/Sc eRF1) recognized UAA and UAG, but not UGA, as stop codons. In the current study, we identified mutations in Eo/Sc eRF1 that restore UGA recognition and define distinct roles for the TASNIKS and YxCxxxF motifs in eRF1 function. Mutations in or near the YxCxxxF motif support the cavity model for stop codon recognition by eRF1. Mutations in the TASNIKS motif eliminated the eRF3 requirement for peptide release at UAA and UAG codons, but not UGA codons. These results suggest that the TASNIKS motif and eRF3 function together to trigger eRF1 conformational changes that couple stop codon recognition and peptide release during eukaryotic translation termination.
使用标准遗传密码的生物体将UAA、UAG和UGA识别为终止密码子，而使用变体密码的物种则经常改变这种终止密码子识别模式。我们之前证明，一种携带八肋游仆虫（Euplotes octocarinatus）结构域1与酿酒酵母（Saccharomyces cerevisiae）结构域2和3融合的杂种eRF1（Eo/Sc eRF1）将UAA和UAG识别为终止密码子，但不将UGA识别为终止密码子。在当前研究中，我们在Eo/Sc eRF1中鉴定出恢复UGA识别的突变，并确定了TASNIKS和YxCxxxF基序在eRF1功能中的不同作用。YxCxxxF基序内或其附近的突变支持eRF1识别终止密码子的空腔模型。TASNIKS基序中的突变消除了eRF3在UAA和UAG密码子处释放肽所需的条件，但在UGA密码子处没有消除。这些结果表明，TASNIKS基序和eRF3共同作用，触发eRF1的构象变化，从而在真核生物翻译终止过程中耦合终止密码子识别和肽释放。
