Poster abstracts

Poster number 55 submitted by Matt Plantinga

Molecular basis of the specificity of the site-specific RNA endonuclease restrictocin

Matthew J. Plantinga (Department of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science), Alexei V. Korennykh (Department of Chemistry, The University of Chicago), Joseph A. Piccirilli (Departments of Chemistry and of Biochemistry and Molecular Biology and The Howard Hughes Medical Institute, The University of Chicago), Carl C. Correll (Department of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science)

Abstract:
Sarcin-like ribotoxins comprise a family of site-specific endoribonucleases that are conserved in both structure and function. Of all the ribosomal phosphodiester bonds, the ribotoxins cleave only one, in the conserved sarcin/ricin loop that folds into a tetraloop and bulged-G motif. Cleavage of a single bond in the tetraloop inhibits translation and ultimately triggers apoptotic cell death. Previously, we showed that cleavage by the sarcin–like ribotoxin restrictocin occurs via two steps: formation of a nonspecific electrostatic complex (E:S) followed by site-recognition and cleavage (NSMB 13, 436 and Biochemistry 46, 12744). Here, we kinetically analyze a series of interface mutants to identify residues that contribute to each step. To identify which residues contribute to binding in the E:S complex we determined the salt dependence of each mutant. Relative to the wild-type salt dependence, only subset of the mutants clustering near but outside of the active site shows significant changes–K110A, K111A and K113A (the lysine triad). Interestingly, the lysine triad also contributes to the subsequent site recognition step. Next, we identified residues that contribute to the 1,000-fold specificity exhibited by restrictocin for the SRL over other substrates. Unexpectedly, mutation of active site residues Y47 and H49, which are expected to interact equally well with any RNA sequence, had no effect on non-specific cleavage but abolished specific cleavage, indicating a role for the active site in specificity. Similarly, mutation of residues forming substrate specific contacts also reduces specific cleavage with little to no effect on non-specific cleavage. Our findings provide support for a model in which electrostatic contacts by the lysine triad position the active site face toward the substrate. Subsequently, the lysine triad contacts the bulged-G motif and active site residues contact the tetraloop of the SRL. Only formation of both sets of contacts permit proper docking of this substrate in the active site to fully engage Y47 and H49 and thereby enhance specific cleavage by 1,000-fold relative to non-specific cleavage.

Keywords: sarcinricin loop, RNA-protein recognition, ribonuclease