Program in Chemistry and Chemical Biology, University of California, San Francisco, California 94158, USA.
RNA. 2011 Feb;17(2):278-90. doi: 10.1261/rna.2382011. Epub 2010 Dec 10.
Cap hydrolysis is a critical step in several eukaryotic mRNA decay pathways and is carried out by the evolutionarily conserved decapping complex containing Dcp2 at the catalytic core. In yeast, Dcp1 is an essential activator of decapping and coactivators such as Edc1 and Edc2 are thought to enhance activity, though their mechanism remains elusive. Using kinetic analysis we show that a crucial function of Dcp1 is to couple the binding of coactivators of decapping to activation of Dcp2. Edc1 and Edc2 bind Dcp1 via its EVH1 proline recognition site and stimulate decapping by 1000-fold, affecting both the K(M) for mRNA and rate of the catalytic step. The C-terminus of Edc1 is necessary and sufficient to enhance the catalytic step, while the remainder of the protein likely increases mRNA binding to the decapping complex. Lesions in the Dcp1 EVH1 domain or the Edc1 proline-rich sequence are sufficient to block stimulation. These results identify a new role of Dcp1, which is to link the binding of coactivators to substrate recognition and activation of Dcp2.
帽水解是几个真核 mRNA 降解途径中的关键步骤，由进化上保守的脱帽复合物完成，该复合物的催化核心含有 Dcp2。在酵母中，Dcp1 是脱帽的必需激活因子，而 Edc1 和 Edc2 等共激活因子被认为可以增强其活性，但它们的作用机制仍然难以捉摸。我们通过动力学分析表明，Dcp1 的一个关键功能是将脱帽共激活因子的结合与 Dcp2 的激活偶联起来。Edc1 和 Edc2 通过其 EVH1 脯氨酸识别位点与 Dcp1 结合，并将脱帽作用提高 1000 倍，影响 mRNA 的 K(M)和催化步骤的速率。Edc1 的 C 末端是增强催化步骤所必需和充分的，而蛋白质的其余部分可能会增加 mRNA 与脱帽复合物的结合。Dcp1 的 EVH1 结构域或 Edc1 富含脯氨酸的序列中的损伤足以阻止这种刺激。这些结果确定了 Dcp1 的一个新作用，即将共激活因子的结合与 Dcp2 的底物识别和激活偶联起来。
