tRNA binding sites of ribosomes from Escherichia coli.
tRNA binding sites of ribosomes from Escherichia coli.
复制标题
大肠杆菌核糖体的 tRNA 结合位点。
作者：
R. Lill;J. M. Robertson;W. Wintermeyer
本文研究了大肠杆菌70S紧偶核糖体中tRNA结合位点的活性和数量。硝酸纤维素过滤和嘌呤霉素测定以直接方式和竞争结合测定的形式使用，后者允许明确测定在tRNA结合中具有活性的核糖体的分数。结果发现，在poly（U）的存在下，活性核糖体结合两个分子的N-AcPhe-tRNAPhe，一个在P和其他在A位点，在Mg 2+浓度为6和20 mM之间。第三个结合位点，除了P和A位点观察脱酰tRNAPhe。在10 mM及以下的Mg2+浓度下，额外位点的占有率非常低。与P和A位点相比，发现tRNA从该位点的解离相当快。这些结果表明，在易位过程中的额外的网站作为出口网站的功能，脱酰tRNA是短暂的结合之前离开核糖体。由于tRNA与该位点的结合不需要poly（U）的存在，因此结合在mRNA固定中的出口位点tRNA的功能似乎不太可能。与同源的tRNAPhe相比，发现来自酵母的异源tRNAPhe与另外的位点结合的亲和力和稳定性都较低。这种差异可能表明了E.大肠杆菌核糖体的tRNA来自同一生物体。
70S tight-couple ribosomes from Escherichia coli were studied with respect to activity and number of tRNA binding sites. The nitrocellulose filtration and puromycin assays were used both in a direct manner and in the form of a competition binding assay, the latter allowing an unambiguous determination of the fraction of ribosomes being active in tRNA binding. It was found that, in the presence of poly(U), the active ribosomes bound two molecules of N-AcPhe-tRNAPhe, one in the P and the other in the A site, at Mg2+ concentrations between 6 and 20 mM. A third binding site in addition to P and A sites was observed for deacylated tRNAPhe. At Mg2+ concentrations of 10 mM and below, the occupancy of the additional site was very low. Dissociation of tRNA from this site was found to be rather fast, as compared to both P and A sites. These results suggest that the additional site during translocation functions as an exit site, to which deacylated tRNA is transiently bound before leaving the ribosome. Since tRNA binding to this site did not require the presence of poly(U), a function of exit site bound tRNA in the fixation of the mRNA appears unlikely. Both the affinity and stability of binding to the additional site were found lower for the heterologous tRNAPhe from yeast as compared to the homologous one. This difference possibly indicates some specificity of the E. coli ribosome for tRNAs from the same organism.