Beringer Malte, Rodnina Marina V
Institute of Physical Biochemistry, University of Witten/Herdecke, Witten, Germany.
Biol Chem. 2007 Jul;388(7):687-91. doi: 10.1515/BC.2007.077.
The major enzymatic activity of the ribosome is the catalysis of peptide bond formation. The active site -- the peptidyl transferase center -- is composed of ribosomal RNA (rRNA), and interactions between rRNA and the reactants, peptidyl-tRNA and aminoacyl-tRNA, are crucial for the reaction to proceed rapidly and efficiently. Here, we describe the influence of rRNA interactions with cytidine residues in A-site substrate analogs (C-puromycin or CC-puromycin), mimicking C74 and C75 of tRNA on the reaction. Base-pairing of C75 with G2553 of 23S rRNA accelerates peptide bond formation, presumably by stabilizing the peptidyl transferase center in its productive conformation. When C74 is also present in the substrate analog, the reaction is slowed down considerably, indicating a slow step in substrate binding to the active site, which limits the reaction rate. The tRNA-rRNA interactions lead to a robust reaction that is insensitive to pH changes or base substitutions in 23S rRNA at the active site of the ribosome.
核糖体的主要酶活性是催化肽键形成。活性位点——肽基转移酶中心——由核糖体RNA（rRNA）组成，rRNA与反应物肽基-tRNA和氨酰-tRNA之间的相互作用对于反应快速高效地进行至关重要。在此，我们描述了rRNA与A位点底物类似物（C-嘌呤霉素或CC-嘌呤霉素）中的胞嘧啶残基相互作用的影响，这些类似物模拟了tRNA的C74和C75对反应的影响。C75与23S rRNA的G2553碱基配对加速了肽键形成，推测是通过将肽基转移酶中心稳定在其有效构象中实现的。当底物类似物中也存在C74时，反应会显著减慢，这表明底物与活性位点结合存在一个缓慢步骤，限制了反应速率。tRNA-rRNA相互作用导致了一个对核糖体活性位点处23S rRNA的pH变化或碱基替换不敏感的稳健反应。
