Abstract
DEAD-box helicases are involved in all steps of RNA metabolism. They are ATP-dependent RNA binding proteins and RNA-dependent ATPases. They can displace short duplexes, but they lack processivity. Their mechanism and functioning are not clearly understood; classical or bulk biochemical assays are not sufficient to answer these questions. Single-molecule techniques provide useful tools, but they are limited in cases where the proteins are nonprocessive and give weak signals. We present here a new, magnetic-tweezers-based, single-molecule assay that is simple and that can sensitively measure the displacement time of a small, hybridized, RNA oligonucleotide. Tens of molecules can be analyzed at the same time. Comparing the displacement times with and without a helicase gives insights into the enzymatic activity of the protein. We used this assay to study yeast Ded1, which is orthologous to human DDX3. Although Ded1 acts on a variety of substrates, we find that Ded1 requires an RNA substrate for its ATP-dependent unwinding activity and that ATP hydrolysis is needed to see this activity. Further, we find that only intramolecular single-stranded RNA extensions enhance this activity. We propose a model where ATP-bound Ded1 stabilizes partially unwound duplexes and where multiple binding events may be needed to see displacement.
Highlights
RNA helicases are ubiquitous proteins that are found in all three kingdoms of life and that are associated with all processes involving RNA, from transcription to decay [1,2,3]
The experimental system was based on manipulating multiple single-stranded DNAs that were attached to a glass slide on one end and magnetic beads on the other [4]
Under sufficient force the DNA could be extended to form a single-stranded chain. This unwound DNA was subsequently stabilized by hybridizing an oligonucleotide of interest at the region corresponding to the apex of the hairpin, which blocked the refolding of the hairpin when the force was reduced
Summary
RNA helicases are ubiquitous proteins that are found in all three kingdoms of life and that are associated with all processes involving RNA, from transcription to decay [1,2,3] Like their DNA helicase counterparts, they are characterized by highly conserved core structures with structural homology to the recombinant protein A (RecA) and that contain highly conserved nucleotide triphosphate binding sites, called the Walker A and B motifs. Most RNA helicases are classified into superfamilies (SFs) 1 and 2, which contain catalytic cores consisting of two, linked, RecA-like domains. Despite their commonly shared cores, these proteins have highly diversified specificities and enzymatic activities. Some are passive and opportunistic: they seem to take advantage of base fraying to progress along the polynucleotide chain
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