Studies of tRNA macro molecular mimicry in translation termination

含有四膜膜结构域1的酵母eRF1结构域交换在体外仅对UGA有反应，并且在体内37℃时不能补充酵母eRF1的缺陷。这一发现表明，来自变异编码生物的eRF1的解码特异性存在于结构域1。然而，野生型eRF1杂种在30℃下完全恢复了eRF1缺陷酵母的生长。四膜虫eRF1在结构域1的尖端含有一个变异序列KATNIKD。杂交eRF1的TASNIKD变体使eRF1无效的酵母存活，尽管在体外实验中，相同的杂交eRF1只对UGA有反应。然而，酵母eRF1携带KATNIKD基序而不是TASNIKS七肽存在于高等真核生物中，在体内仍然是全能的。综上所述，这些数据表明，像四膜虫这样的变异遗传密码生物在体内具有解码三个停止密码子的内在潜力，并且KAT三肽与其他序列在结构域1内的相互作用调节了四膜虫eRF1的解码特异性。嗜热热菌RRF在大肠杆菌中无功能。通过引入表达大肠杆菌EF-G的质粒，使其tRNA-mimic结构域发生表面变化，或者用Thermus结构域取代大肠杆菌EF-G tRNA-mimic结构域，使其发挥功能。Thermus RRF也可以通过在其反密码子臂模拟区引入表面取代来激活。这些功能获得表型依赖于异源EF-G和RRF等位基因的组合。这些突变研究表明，EF-G运动作用通过模拟反密码子臂的结构域之间的特定表面接触传递到RBF。

A yeast eRF1-domain swap containing Tetrahymena domain 1 responded only to UGA in vitro and failed to complement a defect in yeast eRF1 in vivo at 37 degrees C. This finding demonstrates that decoding specificity of eRF1 from variant code organisms resides at domain 1. However, the wild-type eRF1 hybrid fully restored the growth of eRF1-deficient yeast at 30 degrees C. Tetrahymena eRF1 contains a variant sequence, KATNIKD, at the tip of domain 1. The TASNIKD variant of hybrid eRF1 rendered the eRF1-nullified yeast viable, although in an in vitro assay, the same hybrid eRF1 responded only to UGA. Nevertheless, the yeast eRF1 bearing the KATNIKD motif instead of the TASNIKS heptapeptide present in higher eukaryotes remains omnipotent in vivo. Collectively, these data suggest that variant genetic code organisms like Tetrahymena have an intrinsic potential to decode three stop codons in vivo, and that interaction within domain 1 between the KAT tripeptide and other sequences modulates the decoding specificity of Tetrahymena eRF1.Thermus thermophilus RRF is nonfunctional in Escherichia coli. It became functional upon introducing a plasmid expressing E. coli EF-G with surface changes in its tRNA-mimic domain or by replacing the E. coli EF-G tRNA-mimic domain by the Thermus domain. Thermus RRF could also be activated by introducing surface substitutions in its anticodon arm-mimic region. These gain-of-function phenotypes depend on the combination of heterologous EF-G and RRF alleles. These mutational studies suggest that EF-G motor action is transmitted to RBF by specific surface contacts between the domains that mimic the anticodon arm.