Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, United States.
Center for Proteomics and Molecular Therapeutics, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL, United States.
Methods Enzymol. 2022;673:77-101. doi: 10.1016/bs.mie.2022.03.058. Epub 2022 May 14.
The RNA helicase Dhr1 from S. cerevisiae is an essential enzyme required for the assembly of the cytosolic small ribosomal subunit (SSU). A critical feature of the SSU is the central pseudoknot, an RNA fold that organizes the overall architecture of the subunit and connects all four domains of the 18S ribosomal RNA (rRNA). The initial folding of rRNA is guided, in part, by the U3 small nucleolar RNA, which base-pairs with the pre-rRNA in such a way as to preclude premature formation of the central pseudoknot. Thus, the essential role of Dhr1 is the unwinding of U3 from the pre-rRNA to allow folding of the central pseudoknot. Enzymes of the DEAH/RNA helicase A-like (RHA) family, to which Dhr1 belongs, are involved in splicing and ribosome biogenesis. They typically unwind RNA duplexes by translocation along a single strand of RNA in a 3' to 5' direction, driven by ATP hydrolysis. The substrate specificity of these enzymes requires tight regulation of their activity, by restricting access to their substrates, requiring adaptors to recruit them to their substrates and mechanisms of inhibiting and activating their activity. Purified Dhr1 is an active RNA-dependent ATPase with specific unwinding activity. Here, we provide detailed protocols for its purification and assays for its ATPase and unwinding activities.
酿酒酵母的 RNA 解旋酶 Dhr1 是组装细胞质小核糖体亚基（SSU）所必需的酶。SSU 的一个关键特征是中央假结，这是一种组织亚基整体结构并连接 18S 核糖体 RNA（rRNA）的所有四个结构域的 RNA 折叠。rRNA 的初始折叠部分受到 U3 小核仁 RNA 的指导，该 RNA 通过碱基配对与 pre-rRNA 结合，从而防止中央假结过早形成。因此，Dhr1 的重要作用是解开 U3 与 pre-rRNA 的结合，以允许中央假结的折叠。属于 DEAH/RNA 解旋酶 A 样（RHA）家族的 Dhr1 酶参与剪接和核糖体生物发生。它们通常通过在 3' 到 5' 方向沿着 RNA 的单链进行易位来解旋 RNA 双链，由 ATP 水解驱动。这些酶的底物特异性需要通过限制其对底物的访问、需要衔接子将它们募集到其底物上以及抑制和激活其活性的机制来严格调节其活性。纯化的 Dhr1 是一种具有特定解旋活性的 RNA 依赖性 ATP 酶。在这里，我们提供了其纯化和 ATP 酶及解旋活性测定的详细方案。
