Protein unties the pseudoknot: S1-mediated unfolding of RNA higher order structure.

Ribosomal protein Si plays important roles in the translation initiation step of many Escherichia coli mRNAs, particularly those with weak Shine-Dalgarno sequences or structured 5' UTRs, in addition to a variety of cellular processes beyond the ribosome. In all cases, the RNA-binding activity of Si is a central feature of its function. While sequence determinants of Si affinity and many elements of the interactions of Si with simple secondary structures are known, mechanistic details of the protein's interactions with RNAs of more complex secondary and tertiary structure are less understood. Here, we investigate the interaction of Si with the well-characterized H-type pseudoknot of a class-I translational preQ(1) riboswitch as a highly structured RNA model whose conformation and structural dynamics can be tuned by the addition of ligands of varying binding affinity, particularly preQ(1), guanine, and 2,6-diaminopurine. Combining biochemical and single molecule fluorescence approaches, we show that Si preferentially interacts with the less folded form of the pseudoknot and promotes a dynamic, partially unfolded conformation. The ability of Si to unfold the RNA is inversely correlated with the structural stability of the pseudoknot. These mechanistic insights delineate the scope and limitations of Si-chaperoned unfolding of structured RNAs.