Pavlov M Y, Freistroffer D V, Heurgué-Hamard V, Buckingham R H, Ehrenberg M
Department of Molecular Biology, BMC, Uppsala, S-75124, Sweden.
J Mol Biol. 1997 Oct 24;273(2):389-401. doi: 10.1006/jmbi.1997.1324.
The dependence of the rate of ribosomal recycling (from initiation via protein elongation and termination, and then back to initiation) on the concentrations of release factor RF1 and the ribosome recycling factor (RRF) has been studied in vitro. High RF1 concentration was found to reduce the rate of ribosomal recycling and the extent of this reduction depended on stop codon context. The inhibitory effect of high RF1 concentrations can be reversed by a corresponding increase in RRF concentration. This indicates that RF1 and RRF have mutually exclusive and perhaps overlapping binding sites on the ribosome. Addition of release factor RF3 to the translation system abolishes the inhibitory effect of high RF1 concentration and increases the overall rate of ribosome recycling. These data can be explained by a three-step model for termination where the first step is RF1-promoted hydrolysis of peptidyl-tRNA. The second step is an intrinsically slow dissociation of RF1 which is accelerated by RF3. The third step, catalysed by RRF and elongation factor G, leads to mobility of the ribosome on mRNA allowing it to enter a further round of translation. In the absence of RF3, RF1 can re-associate rapidly with the ribosome after peptidyl-tRNA hydrolysis, preventing RRF from entering the ribosomal A-site and thereby inhibiting ribosomal recycling. The overproduction of RF1 in cells deficient in RRF or lacking RF3 has effects on growth rate predicted by the in vitro experiments.
核糖体循环速率（从起始经蛋白质延伸和终止，再回到起始）对释放因子RF1和核糖体循环因子（RRF）浓度的依赖性已在体外进行了研究。发现高浓度的RF1会降低核糖体循环速率，且这种降低的程度取决于终止密码子的上下文。高浓度RF1的抑制作用可通过相应增加RRF浓度来逆转。这表明RF1和RRF在核糖体上具有相互排斥且可能重叠的结合位点。向翻译系统中添加释放因子RF3可消除高浓度RF1的抑制作用，并提高核糖体循环的总体速率。这些数据可用终止的三步模型来解释，其中第一步是RF1促进肽基 - tRNA的水解。第二步是RF1的固有缓慢解离，RF3可加速这一过程。第三步由RRF和延伸因子G催化，导致核糖体在mRNA上移动，使其能够进入新一轮翻译。在没有RF3的情况下，RF1在肽基 - tRNA水解后可迅速与核糖体重新结合，阻止RRF进入核糖体A位点，从而抑制核糖体循环。在缺乏RRF或缺少RF3的细胞中过量表达RF1对生长速率的影响与体外实验预测的一致。
