Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, 630090, Russia.
Nucleic Acids Res. 2011 Sep 1;39(16):7134-46. doi: 10.1093/nar/gkr376. Epub 2011 May 20.
Positioning of release factor eRF1 toward adenines and the ribose-phosphate backbone of the UAAA stop signal in the ribosomal decoding site was studied using messenger RNA (mRNA) analogs containing stop signal UAA/UAAA and a photoactivatable cross-linker at definite locations. The human eRF1 peptides cross-linked to these analogs were identified. Cross-linkers on the adenines at the 2nd, 3rd or 4th position modified eRF1 near the conserved YxCxxxF loop (positions 125-131 in the N domain), but cross-linker at the 4th position mainly modified the tripeptide 26-AAR-28. This tripeptide cross-linked also with derivatized 3'-phosphate of UAA, while the same cross-linker at the 3'-phosphate of UAAA modified both the 26-28 and 67-73 fragments. A comparison of the results with those obtained earlier with mRNA analogs bearing a similar cross-linker at the guanines indicates that positioning of eRF1 toward adenines and guanines of stop signals in the 80S termination complex is different. Molecular modeling of eRF1 in the 80S termination complex showed that eRF1 fragments neighboring guanines and adenines of stop signals are compatible with different N domain conformations of eRF1. These conformations vary by positioning of stop signal purines toward the universally conserved dipeptide 31-GT-32, which neighbors guanines but is oriented more distantly from adenines.
定位释放因子 eRF1 向腺嘌呤和 UAAA 终止信号的核糖-磷酸骨架在核糖体解码位点被研究使用信使 RNA（mRNA）类似物含有终止信号 UAA/UAAA 和一个光活性交联剂在确定的位置。人的 eRF1 肽交联到这些类似物被鉴定。交联剂在腺嘌呤的第 2、3 或 4 位修饰 eRF1 附近保守的 YxCxxxF 环（位置 125-131 在 N 域），但交联剂在第 4 位主要修饰三肽 26-AAR-28。三肽交联也与衍生的 UAA 3′-磷酸，而同样的交联剂在 UAAA 3′-磷酸修饰 26-28 和 67-73 片段。与早些时候用带有类似交联剂的 mRNA 类似物获得的结果进行比较表明，eRF1 在 80S 终止复合物中向终止信号的腺嘌呤和鸟嘌呤的定位是不同的。eRF1 在 80S 终止复合物中的分子建模表明，eRF1 片段相邻的鸟嘌呤和腺嘌呤的终止信号是兼容的不同的 N 域构象的 eRF1。这些构象的变化通过终止信号嘌呤的定位向普遍保守的二肽 31-GT-32，相邻的鸟嘌呤，但定位更远从腺嘌呤。
