Intrinsic dynamics of an extended hydrophobic core in the S. cerevisiae RNase III dsRBD contributes to recognition of specific RNA binding sites.

ABSTRACT: The Saccharomyces cerevisiae RNase III enzyme Rnt1p preferentially binds to double-stranded RNA hairpin substrates with a conserved (A/u)GNN tetraloop fold, via shape-specific interactions by its double-stranded RNA-binding domain (dsRBD) helix ?1 to the tetraloop minor groove. To investigate whether conformational flexibility in the dsRBD regulates the binding specificity, we determined the backbone dynamics of the Rnt1p dsRBD in the free and AGAA hairpin-bound states using NMR spin-relaxation experiments. The intrinsic microsecond-to-millisecond timescale dynamics of the dsRBD suggests that helix ?1 undergoes conformational sampling in the free state, with large dynamics at some residues in the ?1-?1 loop (?1-?1 hinge). To correlate free dsRBD dynamics with structural changes upon binding, we determined the solution structure of the free dsRBD used in the previously determined RNA-bound structures. The Rnt1p dsRBD has an extended hydrophobic core comprising helix ?1, the ?1-?1 loop, and helix ?3. Analysis of the backbone dynamics and structures of the free and bound dsRBD reveals that slow-timescale dynamics in the ?1-?1 hinge are associated with concerted structural changes in the extended hydrophobic core that govern binding of helix ?1 to AGAA tetraloops. The dynamic behavior of the dsRBD bound to a longer AGAA hairpin reveals that dynamics within the hydrophobic core differentiate between specific and nonspecific sites. Mutations of residues in the ?1-?1 hinge result in changes to the dsRBD stability and RNA-binding affinity and cause defects in small nucleolar RNA processing invivo. These results reveal that dynamics in the extended hydrophobic core are important for binding site selection by the Rnt1p dsRBD.

SUBMITTER: Hartman E 

PROVIDER: S-EPMC3557707 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature