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.

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Journal Article

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Lin R, Correll CC, Johnson AW

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