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DEAD-box proteins are the largest family of nucleic acid helicases and are crucial to RNA metabolism throughout all domains of life1,2. They contain a conserved ‘helicase core’ of two RecA-like domains (domains 1 and 2; D1 and D2, respectively), which uses ATP to catalyze the unwinding of short RNA duplexes by nonprocessive, local strand separation3. This mode of action differs from that of translocating helicases and allows DEAD-box proteins to remodel large RNAs and RNA-protein complexes without globally disrupting RNA structure4. However, the structural basis for this distinctive mode of RNA-unwinding remains unclear. Here, structural, biochemical, and genetic analyses of the yeast DEAD-box protein Mss116p indicate that the helicase core domains have modular functions that enable a novel mechanism for RNA duplex recognition and unwinding. By investigating D1 and D2 individually and together, we find that D1 acts as an ATP-binding domain and D2 functions as an RNA-duplex recognition domain. D2 contains a nucleic acid-binding pocket that is formed by conserved DEAD-box protein sequence motifs and accommodates A-form but not B-form duplexes, providing a basis for RNA substrate specificity. Upon a conformational change in which the two core domains join to form a ‘closed-state’ with an ATPase active site, conserved motifs in D1 promote the unwinding of duplex substrates bound to D2 by excluding one RNA strand and bending the other. Our results provide a comprehensive structural model for how DEAD-box proteins recognize and unwind RNA duplexes. This model explains key features of DEAD-box protein function and affords new perspective on how the evolutionarily related cores of other RNA and DNA helicases diverged to use different mechanisms.

Topological knots are found in a considerable number of protein structures, but it is not clear how they knot and fold within the cellular environment. We investigated the behavior of knotted protein molecules as they are first synthesized by the ribosome using a cell-free translation system. We found that newly translated knotted proteins can spontaneously self-tie and do not require the assistance of molecular chaperones to fold correctly to their trefoil-knotted structures. This process is slow but efficient, and we found no evidence of misfolded species. A kinetic analysis indicates that the knotting process is rate limiting, occurs post-translationally, and is specifically and significantly (P < 0.001) accelerated by the GroEL-GroES chaperonin complex. This demonstrates a new active mechanism for this molecular chaperone and suggests that chaperonin-catalyzed knotting probably dominates in vivo. These results explain how knotted protein structures have withstood evolutionary pressures despite their topological complexity.

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