Dabrowski M, Junemann R, Schafer M A, Spahn C M, Nierhaus K H, Alekseeva E V, Dontsova O A, Shpanchenko O V, Bogdanov A A
Biokhimiia. 1996 Nov;61(11):1971-83.
A novel footprinting method was recently developed which identifies phosphate groups of RNA involved in strong RNA-RNA and RNA-protein interactions. The method is based on iodine-dependent RNA cleavage at phosphothioate groups as long as these groups are not protected from iodine. Our recent studies of mRNA and tRNA regions protected in active ribosomes are summarized; initiation state of ribosomes as well as two elongation states in pre- and post-translocational states were analyzed. Only one phosphate group of mRNA, which was two positions upstream of the decoding codons, was weakly protected in longation complexes, whereas this group and the phosphate groups in the Shine-Dalgarno sequence were protected in the initiation complex. No protection was observed downstream of the decoding codons. On the contrary, numerous phosphate residues of tRNA were protected by the ribosome. The tRNA protection patterns significantly varied between two tRNAs simultaneously bound to the ribosome. The protection pattern of an individual tRNA was not significantly affected by translocation. The data indicate that both tRNA molecules are tightly bound to the ribosome, whereas mRNA is fixed predominantly by two tRNAs via codon-anticodon interaction. A possible translocation mechanism is suggested.
最近开发了一种新的足迹法，可识别参与强RNA-RNA和RNA-蛋白质相互作用的RNA磷酸基团。该方法基于硫代磷酸酯基团处碘依赖性RNA切割，前提是这些基团未受到碘的保护。总结了我们最近对活性核糖体中受保护的mRNA和tRNA区域的研究；分析了核糖体的起始状态以及转位前和转位后状态下的两种延伸状态。在延伸复合物中，只有位于解码密码子上游两个位置的一个mRNA磷酸基团受到弱保护，而该基团以及Shine-Dalgarno序列中的磷酸基团在起始复合物中受到保护。在解码密码子下游未观察到保护作用。相反，tRNA的许多磷酸残基受到核糖体的保护。同时结合到核糖体上的两种tRNA之间的tRNA保护模式有显著差异。单个tRNA的保护模式不受转位的显著影响。数据表明，两个tRNA分子都紧密结合到核糖体上，而mRNA主要通过密码子-反密码子相互作用由两个tRNA固定。提出了一种可能的转位机制。
