Koch Miriam, Clementi Nina, Rusca Nicola, Vögele Paul, Erlacher Matthias, Polacek Norbert
a Department of Chemistry and Biochemistry; University of Bern ; Bern , Switzerland.
RNA Biol. 2015;12(1):70-81. doi: 10.1080/15476286.2015.1017218.
During the elongation cycle of protein biosynthesis, tRNAs traverse through the ribosome by consecutive binding to the 3 ribosomal binding sites (A-, P-, and E- sites). While the ribosomal A- and P-sites have been functionally well characterized in the past, the contribution of the E-site to protein biosynthesis is still poorly understood in molecular terms. Previous studies suggested an important functional interaction of the terminal residue A76 of E-tRNA with the nucleobase of the universally conserved 23S rRNA residue C2394. Using an atomic mutagenesis approach to introduce non-natural nucleoside analogs into the 23S rRNA, we could show that removal of the nucleobase or the ribose 2'-OH at C2394 had no effect on protein synthesis. On the other hand, our data disclose the importance of the highly conserved E-site base pair G2421-C2395 for effective translation. Ribosomes with a disrupted G2421-C2395 base pair are defective in tRNA binding to the E-site. This results in an impaired translation of genuine mRNAs, while homo-polymeric templates are not affected. Cumulatively our data emphasize the importance of E-site tRNA occupancy and in particular the intactness of the 23S rRNA base pair G2421-C2395 for productive protein biosynthesis.
在蛋白质生物合成的延伸循环中，转运RNA（tRNA）通过依次结合核糖体的3个结合位点（A位点、P位点和E位点）在核糖体中穿梭。虽然过去核糖体的A位点和P位点在功能上已得到充分表征，但从分子角度来看，E位点对蛋白质生物合成的贡献仍知之甚少。先前的研究表明，空载tRNA（E-tRNA）的末端残基A76与普遍保守的23S核糖体RNA（rRNA）残基C2394的核碱基存在重要的功能相互作用。通过原子诱变方法将非天然核苷类似物引入23S rRNA，我们发现去除C2394处的核碱基或核糖2'-羟基对蛋白质合成没有影响。另一方面，我们的数据揭示了高度保守的E位点碱基对G2421-C2395对有效翻译的重要性。具有破坏的G2421-C2395碱基对的核糖体在tRNA与E位点的结合方面存在缺陷。这导致天然信使核糖核酸（mRNA）的翻译受损，而均聚物模板不受影响。总的来说，我们的数据强调了E位点tRNA占据的重要性，特别是23S rRNA碱基对G2421-C2395的完整性对高效蛋白质生物合成的重要性。
