Innsbruck Biocenter, Medical University Innsbruck, Division of Genomics and RNomics, Innsbruck, Austria.
Nucleic Acids Res. 2010 Aug;38(14):4844-55. doi: 10.1093/nar/gkq213. Epub 2010 Apr 7.
Despite the fact that all 23S rRNA nucleotides that build the ribosomal peptidyl transferase ribozyme are universally conserved, standard and atomic mutagenesis studies revealed the nucleobase identities being non-critical for catalysis. This indicates that these active site residues are highly conserved for functions distinct from catalysis. To gain insight into potential contributions, we have manipulated the nucleobases via an atomic mutagenesis approach and have utilized these chemically engineered ribosomes for in vitro translation reactions. We show that most of the active site nucleobases could be removed without significant effects on polypeptide production. Our data however highlight the functional importance of the universally conserved non-Watson-Crick base pair at position A2450-C2063. Modifications that disrupt this base pair markedly impair translation activities, while having little effects on peptide bond formation, tRNA drop-off and ribosome-dependent EF-G GTPase activity. Thus it seems that disruption of the A2450-C2063 pair inhibits a reaction following transpeptidation and EF-G action during the elongation cycle. Cumulatively our data are compatible with the hypothesis that the integrity of this A-C wobble base pair is essential for effective tRNA translocation through the peptidyl transferase center during protein synthesis.
尽管构建核糖体肽基转移酶核酶的 23S rRNA 核苷酸全部都普遍保守，但标准和原子突变研究表明，核碱基的身份对于催化并非关键。这表明这些活性位点残基高度保守，具有不同于催化的功能。为了深入了解潜在的贡献，我们通过原子突变方法对核碱基进行了操作，并利用这些化学工程化的核糖体进行体外翻译反应。我们表明，大多数活性位点核碱基可以被去除，而对多肽产生没有显著影响。然而，我们的数据突出了普遍保守的非 Watson-Crick 碱基对 A2450-C2063 位置的功能重要性。破坏该碱基对的修饰显著削弱了翻译活性，而对肽键形成、tRNA 脱落和核糖体依赖性 EF-G GTP 酶活性几乎没有影响。因此，似乎破坏 A2450-C2063 对会抑制转肽反应和延伸循环中 EF-G 作用后的反应。总的来说，我们的数据与以下假设一致，即该 A-C 摆动碱基对的完整性对于蛋白质合成过程中 tRNA 有效通过肽基转移酶中心的易位是必不可少的。
