Studies of tRNA macro molecular mimicry in translation termination

真核生物的翻译终止是由两个相互作用的释放因子eRF1和eRF3控制的。在三种状态（游离蛋白、GDP-和gtp结合形式）下测定的S. pombe eRF3的eef1样区域的晶体结构显示出与EF-Tu相似的总体结构，尽管结构域排列完全不同。与EF-Tu相比，GDP/GTP结合eRF3c不会引起剧烈的构象变化，并且GDP结合eRF3c不需要Mg2。高浓度的Mg2加速了GDP的释放，这表明eRF3与其他gtpase的核苷酸交换机制存在新的机制。将序列保守定位到分子表面，结合诱变分析，确定了eRF1结合区，揭示了eRF3 C端的重要功能。n端延伸，富含酸性氨基酸，阻断了eRF1结合位点，可能以竞争方式调节eRF1与eRF3的结合。核糖体再循环因子（RRF）与延伸因子EF-G一起分解终止后的核糖体复合物，释放核糖体进行新一轮多肽合成。RRF如何与EF-G相互作用并分解终止后核糖体尚不清楚。RRF在结构上与tRNA相似，因此被认为与核糖体A位点结合，并在核糖体拆卸过程中被EF-G作为tRNA的模拟物而易位。然而，在GTP水解中保持活性但在tRNA易位中存在缺陷的EF-G变体在体内和体外完全激活RRF功能。此外，RRF和GTP形式的EF-G在体外不共占据终止核糖体；rf由EF-G从预形成的络合物中喷射出来。这些发现表明，RRF不是tRNA的功能模拟物，并且独立于EF-G的易位活性来拆卸终止后的核糖体复合物。

Translation termination in eukaryotes is governed by two interacting release factors, eRF1 and eRF3. The crystal structure of the eEF1-like region of eRF3 from S. pombe determined in three states (free protein, GDP-, and GTP-bound forms) reveals an overall structure that is similar to EF-Tu, although with quite different domain arrangements. In contrast to EF-Tu, GDP/GTP binding to eRF3c does not induce dramatic conformational changes, and Mg2 is not required for GDP binding to eRF3c. Mg2 at higher concentration accelerates GDP release, suggesting a novel mechanism for nucleotide exchange on eRF3 from that of other GTPases. Mapping sequence conservation onto the molecular surface, combined with mutagenesis analysis, identified the eRF1 binding region, and revealed an essential function for the C terminus of eRF3. The N-terminal extension, rich in acidic amino acids, blocks the proposed eRF1 binding site, potentially regulating eRF1 binding to eRF3 in a competitive manner.Ribosome recycling factor (RRF) disassembles post termination ribosomal complexes in concert with elongation factor EF-G freeing the ribosome for a new round of polypeptide synthesis. How RRF interacts with EF-G and disassembles post-termination ribosomes is unknown. RRF is structurally similar to tRNA and is therefore thought to bind to the ribosomal A site and be translocated by EF-G during ribosome disassembly as a mimic of tRNA. However, EF-G variants that remain active in GTP hydrolysis but are defective in tRNA translocation fully activate RRF function in vivo and in vitro. Furthermore, RRF and the GTP form of EF-G do not co-occupy the terminating ribosome in vitro ; RRF is ejected by EF-G from the preformed complex. These findings suggest that RRF is not a functional mimic of tRNA and disassembles the post-termination ribosomal complex indepen dently of the translocation activity of EF-G.