The role of the universally conserved A2450-C2063 base pair in the ribosomal peptidyl transferase center.
The role of the universally conserved A2450-C2063 base pair in the ribosomal peptidyl transferase center.
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DOI：
10.1093/nar/gkq213
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发表时间：
2010-08
影响因子：
14.9
通讯作者：
Polacek N
中科院分区：
文献类型：
--
作者：
Chirkova A;Erlacher MD;Clementi N;Zywicki M;Aigner M;Polacek N
尽管构建核糖体肽基转移酶核酶的所有23 S rRNA核苷酸都是普遍保守的，但标准和原子诱变研究揭示了核碱基身份对于催化是非关键的。这表明这些活性位点残基对于不同于催化的功能是高度保守的。为了深入了解潜在的贡献，我们通过原子诱变方法操纵核碱基，并利用这些化学工程核糖体进行体外翻译反应。我们表明，大多数的活性位点的核碱基可以被删除，而不会对多肽的生产产生显着的影响。然而，我们的数据突出了普遍保守的非沃森-克里克碱基对在位置A2450-C2063的功能重要性。破坏该碱基对的修饰显著损害翻译活性，而对肽键形成、tRNA脱落和核糖体依赖性EF-G GTdase活性几乎没有影响。因此，似乎A2450-C2063对的破坏抑制了在延伸周期期间转肽作用和EF-G作用之后的反应。累积我们的数据是兼容的假设，这A-C摆动碱基对的完整性是必不可少的有效的tRNA易位通过肽基转移酶中心在蛋白质合成。
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.
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