Erlacher Matthias D, Lang Kathrin, Shankaran Nisha, Wotzel Brigitte, Hüttenhofer Alexander, Micura Ronald, Mankin Alexander S, Polacek Norbert
Innsbruck Biocenter, Division of Genomics and RNomics-Innsbruck Medical University Fritz-Pregl Strasse 3, 6020 Innsbruck, Austria.
Nucleic Acids Res. 2005 Mar 14;33(5):1618-27. doi: 10.1093/nar/gki308. Print 2005.
The main enzymatic reaction of the large ribosomal subunit is peptide bond formation. Ribosome crystallography showed that A2451 of 23S rRNA makes the closest approach to the attacking amino group of aminoacyl-tRNA. Mutations of A2451 had relatively small effects on transpeptidation and failed to unequivocally identify the crucial functional group(s). Here, we employed an in vitro reconstitution system for chemical engineering the peptidyl transferase center by introducing non-natural nucleosides at position A2451. This allowed us to investigate the peptidyl transfer reaction performed by a ribosome that contained a modified nucleoside at the active site. The main finding is that ribosomes carrying a 2'-deoxyribose at A2451 showed a compromised peptidyl transferase activity. In variance, adenine base modifications and even the removal of the entire nucleobase at A2451 had only little impact on peptide bond formation, as long as the 2'-hydroxyl was present. This implicates a functional or structural role of the 2'-hydroxyl group at A2451 for transpeptidation.
大核糖体亚基的主要酶促反应是肽键形成。核糖体晶体学显示，23S rRNA的A2451与氨酰-tRNA的进攻氨基距离最近。A2451的突变对转肽作用的影响相对较小，并且未能明确鉴定出关键功能基团。在此，我们采用了一种体外重组系统，通过在A2451位点引入非天然核苷来对肽基转移酶中心进行化学工程改造。这使我们能够研究在活性位点含有修饰核苷的核糖体所进行的肽基转移反应。主要发现是，在A2451位点携带2'-脱氧核糖的核糖体表现出受损的肽基转移酶活性。与此不同的是，只要2'-羟基存在，腺嘌呤碱基修饰甚至A2451位点整个核苷酸碱基的去除对肽键形成的影响都很小。这表明A2451位点的2'-羟基基团在转肽作用中具有功能或结构作用。
