(A) (A) RNA-free Rnt1p medium-dsRBD structure in the same orientations used in the surface representations. (B) A region of positive electrostatic potential on the surface of the Rnt1p dsRBD crystal structure (blue patches) coincides with the RNA-binding surface of the protein. (C) Residues that shift upon RNA binding identify the RNA-binding surface of Rnt1p (red-coded residues). (D) Residues located close to the AGGA tetraloop as identified in the spin-labeling experiments (green-coded residues). The tetraloop interaction site is precisely determined by the spin label experiment and coincides with helix _1, loop 1 and the C-terminus of helix _3.

Rnt1 endoribonuclease, the yeast homolog of RNAse III, plays an important role in the maturation of a diverse set of RNAs. The enzymatic activity requires a conserved catalytic domain, while RNA binding requires the double-stranded RNA-binding domain (dsRBD) at the C-terminus of the protein. While bacterial RNAse III enzymes cleave double-stranded RNA, Rnt1p specifically cleaves RNAs that possess short irregular stem-loops containing 12_14 base pairs interrupted by internal loops and bulges and capped by conserved AGNN tetraloops. Consistent with this substrate specificity, the isolated Rnt1p dsRBD and the 30_40 amino acids that follow bind to AGNN-containing stem-loops preferentially in vitro. In order to understand how Rnt1p recognizes its cognate processing sites, we have defined its minimal RNA-binding domain and determined its structure by solution NMR spectroscopy and X-ray crystallography. We observe a new carboxy-terminal helix following a canonical dsRBD structure. Removal of this helix reduces binding to Rnt1p substrates. The results suggest that this helix allows the Rnt1p dsRBD to bind to short RNA stem-loops by modulating the conformation of helix 1, a key RNA-recognition element of the dsRBD.