Petropoulos Alexandros D, McDonald Megan E, Green Rachel, Zaher Hani S
From the Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 and.
the Department of Biology, Washington University in St. Louis, St. Louis, Missouri 63130
J Biol Chem. 2014 Jun 20;289(25):17589-96. doi: 10.1074/jbc.M114.564989. Epub 2014 May 5.
In bacteria, stop codons are recognized by two similar class 1 release factors, release factor 1 (RF1) and release factor 2 (RF2). Normally, during termination, the class 2 release factor 3 (RF3), a GTPase, functions downstream of peptide release where it accelerates the dissociation of RF1/RF2 prior to ribosome recycling. In addition to their canonical function in termination, both classes of release factor are also involved in a post peptidyl transfer quality control (post PT QC) mechanism where the termination factors recognize mismatched (i.e. error-containing) ribosome complexes and promote premature termination. Here, using a well defined in vitro system, we explored the role of release factors in canonical termination and post PT QC. As reported previously, during canonical termination, RF1 and RF2 recognize stop codons in a similar manner, and RF3 accelerates their rate of dissociation. During post PT QC, only RF2 (and not RF1) effectively binds to mismatched ribosome complexes; and whereas the addition of RF3 to RF2 increased its rate of release on mismatched complexes, the addition of RF3 to RF1 inhibited its rate of release but increased the rate of peptidyl-tRNA dissociation. Our data strongly suggest that RF2, in addition to its primary role in peptide release, functions as the principle factor for post PT QC.
在细菌中，终止密码子由两个相似的1类释放因子识别，即释放因子1（RF1）和释放因子2（RF2）。通常，在终止过程中，2类释放因子3（RF3）作为一种GTP酶，在肽释放的下游发挥作用，在核糖体循环之前加速RF1/RF2的解离。除了它们在终止过程中的典型功能外，这两类释放因子还参与了肽基转移后质量控制（post PT QC）机制，在该机制中，终止因子识别错配（即含错误）的核糖体复合物并促进过早终止。在这里，我们使用一个定义明确的体外系统，探索了释放因子在典型终止和post PT QC中的作用。如先前报道，在典型终止过程中，RF1和RF2以相似的方式识别终止密码子，并且RF3加速它们的解离速率。在post PT QC过程中，只有RF2（而不是RF1）能有效地结合错配的核糖体复合物；虽然向RF2中添加RF3会增加其在错配复合物上的释放速率，但向RF1中添加RF3会抑制其释放速率，但会增加肽基-tRNA的解离速率。我们的数据强烈表明，RF2除了在肽释放中的主要作用外，还作为post PT QC的主要因子发挥作用。
