Sergiev Petr V, Lesnyak Dmitry V, Kiparisov Sergey V, Burakovsky Dmitry E, Leonov Andrei A, Bogdanov Alexey A, Brimacombe Richard, Dontsova Olga A
Department of Chemistry and A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, 119899, Russia.
Nucleic Acids Res. 2005 Oct 20;33(18):6048-56. doi: 10.1093/nar/gki910. Print 2005.
Ribosomes synthesize proteins according to the information encoded in mRNA. During this process, both the incoming amino acid and the nascent peptide are bound to tRNA molecules. Three binding sites for tRNA in the ribosome are known: the A-site for aminoacyl-tRNA, the P-site for peptidyl-tRNA and the E-site for the deacylated tRNA leaving the ribosome. Here, we present a study of Escherichia coli ribosomes with the E-site binding destabilized by mutation C2394G of the 23S rRNA. Expression of the mutant 23S rRNA in vivo caused increased frameshifting and stop codon readthrough. The progression of these ribosomes through the ribosomal elongation cycle in vitro reveals ejection of deacylated tRNA during the translocation step or shortly after. E-site compromised ribosomes can undergo translocation, although in some cases it is less efficient and results in a frameshift. The mutation affects formation of the P/E hybrid site and leads to a loss of stimulation of the multiple turnover GTPase activity of EF-G by deacylated tRNA bound to the ribosome.
核糖体根据mRNA中编码的信息合成蛋白质。在此过程中，进入的氨基酸和新生肽都与tRNA分子结合。核糖体中tRNA有三个结合位点：氨酰tRNA的A位点、肽酰tRNA的P位点和离开核糖体的脱酰tRNA的E位点。在此，我们展示了一项对大肠杆菌核糖体的研究，其E位点结合因23S rRNA的C2394G突变而不稳定。突变型23S rRNA在体内的表达导致移码增加和终止密码子通读。这些核糖体在体外核糖体延伸循环中的进展揭示了在转位步骤期间或之后不久脱酰tRNA的排出。E位点受损的核糖体可以进行转位，尽管在某些情况下效率较低并导致移码。该突变影响P/E杂交位点的形成，并导致与核糖体结合的脱酰tRNA对EF-G的多轮GTPase活性的刺激丧失。
