Spahn C M, Schäfer M A, Krayevsky A A, Nierhaus K H
Max-Planck-Institut für Molekulare Genetik, AG Ribosomen, Ihnestrasse 73, D-14195 Berlin, Germany.
J Biol Chem. 1996 Dec 20;271(51):32857-62. doi: 10.1074/jbc.271.51.32857.
Two nucleotides of the 23 S rRNA gene were mutated; the nucleotides correspond to the first two positions of the universally conserved sequence PsiGG2582 at the peptidyltransferase ring of 23 S rRNA. The ribosomes containing the altered 23 S rRNA were analyzed. Previously, it was shown that ribosomal assembly was indistinguishable from that in wild-type cells, that the flow of the corresponding 50 S subunit into the polysome fraction was not restricted, but that the ribosomes were strongly impaired in poly(Phe) synthesis (C. M. T. Spahn, J. Remme, M. A. Schäfer, and K. H. Nierhaus (1996) J. Biol. Chem. 271, 32849-32856). Here we apply assay systems exclusively testing the puromycin reaction of ribosomes carrying plasmid-born rRNA, a dipeptide assay using the minimal P site donor pA(fMet) and a translocation system not depending on the puromycin reaction. The mutations in helix 90 exclusively abolish or severely impair the ribosome capability to catalyze AcPhe-puromycin formation. A possible explanation of these observations is that G2581 and Psi2580 (and possibly also G2582) are part of the binding site of C75 of peptidyl-tRNA in the P site. The results suggest that in this case, however, such an interaction would disobey canonical base pairing.
23 S rRNA基因的两个核苷酸发生了突变；这些核苷酸对应于23 S rRNA肽基转移酶环上普遍保守序列PsiGG2582的前两个位置。对含有改变后的23 S rRNA的核糖体进行了分析。此前研究表明，核糖体组装与野生型细胞中的组装没有区别，相应的50 S亚基向多核糖体部分的流动不受限制，但核糖体在多聚（苯丙氨酸）合成中受到严重损害（C.M.T. Spahn、J. Remme、M.A. Schäfer和K.H. Nierhaus（1996年）《生物化学杂志》271卷，32849 - 32856页）。在此，我们应用专门测试携带质粒来源rRNA的核糖体嘌呤霉素反应的检测系统、使用最小P位点供体pA（fMet）的二肽检测以及不依赖嘌呤霉素反应的转位系统。90螺旋中的突变专门消除或严重损害了核糖体催化AcPhe - 嘌呤霉素形成的能力。对这些观察结果的一种可能解释是，G2581和Psi2580（可能还有G2582）是P位点肽基 - tRNA的C75结合位点的一部分。然而，结果表明，在这种情况下，这种相互作用将不符合经典碱基配对规则。
