Chen Yuanwei, Kaji Akira, Kaji Hideko, Cooperman Barry S
Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA.
Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Nucleic Acids Res. 2017 Sep 29;45(17):10168-10177. doi: 10.1093/nar/gkx694.
Bacterial ribosome recycling requires breakdown of the post-termination complex (PoTC), comprising a messenger RNA (mRNA) and an uncharged transfer RNA (tRNA) cognate to the terminal mRNA codon bound to the 70S ribosome. The translation factors, elongation factor G and ribosome recycling factor, are known to be required for recycling, but there is controversy concerning whether these factors act primarily to effect the release of mRNA and tRNA from the ribosome, with the splitting of the ribosome into subunits being somewhat dispensable, or whether their main function is to catalyze the splitting reaction, which necessarily precedes mRNA and tRNA release. Here, we utilize three assays directly measuring the rates of mRNA and tRNA release and of ribosome splitting in several model PoTCs. Our results largely reconcile these previously held views. We demonstrate that, in the absence of an upstream Shine-Dalgarno (SD) sequence, PoTC breakdown proceeds in the order: mRNA release followed by tRNA release and then by 70S splitting. By contrast, in the presence of an SD sequence all three processes proceed with identical apparent rates, with the splitting step likely being rate-determining. Our results are consistent with ribosome profiling results demonstrating the influence of upstream SD-like sequences on ribosome occupancy at or just before the mRNA stop codon.
细菌核糖体循环需要分解终止后复合物（PoTC），该复合物由信使核糖核酸（mRNA）和与结合在70S核糖体上的末端mRNA密码子同源的无负载转运核糖核酸（tRNA）组成。已知翻译因子延伸因子G和核糖体循环因子是核糖体循环所必需的，但关于这些因子主要是作用于使mRNA和tRNA从核糖体上释放，而核糖体亚基的解离在某种程度上是次要的，还是其主要功能是催化必然先于mRNA和tRNA释放的解离反应，存在争议。在这里，我们利用三种测定方法直接测量了几种模型PoTC中mRNA和tRNA释放以及核糖体解离的速率。我们的结果在很大程度上调和了这些先前的观点。我们证明，在没有上游夏因-达尔加诺（SD）序列的情况下，PoTC的分解按以下顺序进行：mRNA释放，接着是tRNA释放，然后是70S核糖体解离。相比之下，在有SD序列的情况下，所有这三个过程以相同的表观速率进行，其中解离步骤可能是限速步骤。我们的结果与核糖体分析结果一致，该结果证明了上游类SD序列对mRNA终止密码子处或之前核糖体占据情况的影响。
