Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093, USA.
Nucleic Acids Res. 2010 Jul;38(12):4108-19. doi: 10.1093/nar/gkq151. Epub 2010 Mar 9.
Ribotoxins are potent inhibitors of protein biosynthesis and inactivate ribosomes from a variety of organisms. The ribotoxin alpha-sarcin cleaves the large 23S ribosomal RNA (rRNA) at the universally conserved sarcin-ricin loop (SRL) leading to complete inactivation of the ribosome and cellular death. The SRL interacts with translation factors that hydrolyze GTP, and it is important for their binding to the ribosome, but its precise role is not yet understood. We studied the effect of alpha-sarcin on defined steps of translation by the bacterial ribosome. alpha-Sarcin-treated ribosomes showed no defects in mRNA and tRNA binding, peptide-bond formation and sparsomycin-dependent translocation. Cleavage of SRL slightly affected binding of elongation factor Tu ternary complex (EF-TuGTPtRNA) to the ribosome. In contrast, the activity of elongation factor G (EF-G) was strongly impaired in alpha-sarcin-treated ribosomes. Importantly, cleavage of SRL inhibited EF-G binding, and consequently GTP hydrolysis and mRNA-tRNA translocation. These results suggest that the SRL is more critical in EF-G than ternary complex binding to the ribosome implicating different requirements in this region of the ribosome during protein elongation.
核糖体毒素是蛋白质生物合成的有效抑制剂，能使来自各种生物体的核糖体失活。α-鹅膏蕈碱内切酶能够在保守的 sarcin-ricin 环（SRL）处切割大 23S 核糖体 RNA（rRNA），导致核糖体完全失活和细胞死亡。SRL 与水解 GTP 的翻译因子相互作用，对于它们与核糖体的结合非常重要，但它的确切作用尚不清楚。我们通过细菌核糖体研究了 α-鹅膏蕈碱内切酶对翻译特定步骤的影响。α-鹅膏蕈碱内切酶处理的核糖体在 mRNA 和 tRNA 结合、肽键形成和斯巴醇依赖性易位方面没有缺陷。SRL 的切割稍微影响了延伸因子 Tu 三元复合物（EF-TuGTPtRNA）与核糖体的结合。相比之下，α-鹅膏蕈碱内切酶处理的核糖体中延伸因子 G（EF-G）的活性受到强烈抑制。重要的是，SRL 的切割抑制了 EF-G 的结合，从而抑制了 GTP 水解和 mRNA-tRNA 易位。这些结果表明，SRL 在 EF-G 结合中比在三元复合物与核糖体结合中更为关键，这表明在蛋白质延伸过程中核糖体的这一区域有不同的要求。
