Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia.
Biochimie. 2010 Jul;92(7):820-5. doi: 10.1016/j.biochi.2010.02.031. Epub 2010 Mar 3.
The eukaryotic ribosomal protein S15 is a key component of the decoding site in contrast to its prokaryotic counterpart, S19p, which is located away from the mRNA binding track on the ribosome. Here, we determined the oligopeptide of S15 neighboring the A site mRNA codon on the human 80S ribosome with the use of mRNA analogues bearing perfluorophenyl azide-modified nucleotides in the sense or stop codon targeted to the 80S ribosomal A site. The protein was cross-linked to mRNA analogues in specific ribosomal complexes that were obtained in the presence of eRF1 in the experiments with mRNAs bearing stop codon. Digestion of modified S15 with various specific proteolytic agents followed by identification of the resulting modified oligopeptides showed that cross-link was in C-terminal fragment in positions 131-145, most probably, in decapeptide 131-PGIGATHSSR-140. The position of cross-linking site on the S15 protein did not depend on the nature of the A site-bound codon (sense or stop codon) and on the presence of polypeptide chain release factor eRF1 in the ribosomal complexes with mRNA analogues bearing a stop codon. The results indicate an involvement of the mentioned decapeptide in the formation of the ribosomal decoding site during elongation and termination of translation. Alignment of amino acid sequences of eukaryotic S15 and its prokaryotic counterpart, S19p from eubacteria and archaea, revealed that decapeptide PGIGATHSSR in positions 131-140 is strongly conserved in eukaryotes and has minor variations in archaea but has no homology with any sequence in C-terminal part of eubacterial S19p, which suggests involvement of the decapeptide in the translation process in a eukaryote-specific manner.
真核核糖体蛋白 S15 是解码位点的关键组成部分，与其原核对应物 S19p 形成对比，后者位于核糖体上的 mRNA 结合轨道之外。在这里，我们使用在有意义或终止密码子上带有全氟苯甲酰叠氮修饰核苷酸的 mRNA 类似物，在存在 eRF1 的情况下，确定了人类 80S 核糖体上 A 位点 mRNA 密码子附近的 S15 寡肽。在带有终止密码子的 mRNA 的实验中，与核糖体复合物中的 mRNA 类似物进行蛋白交联，然后用各种特定的蛋白水解酶消化修饰的 S15，再鉴定得到的修饰寡肽，结果表明交联发生在 C 端片段 131-145 位，很可能是在十肽 131-PGIGATHSSR-140 中。交联位置在 S15 蛋白上不依赖于 A 位结合密码子（有意义或终止密码子）的性质，也不依赖于在含有带有终止密码子的 mRNA 类似物的核糖体复合物中多肽链释放因子 eRF1 的存在。结果表明，在翻译延伸和终止过程中，上述十肽参与了核糖体解码位点的形成。真核 S15 和其原核对应物 S19p（来自真细菌和古细菌）的氨基酸序列比对表明，131-140 位的十肽 PGIGATHSSR 在真核生物中高度保守，在古细菌中略有变化，但与真细菌 S19p 的 C 端部分的任何序列都没有同源性，这表明该十肽以真核生物特有的方式参与翻译过程。
