The Ribotoxin Restrictocin Recognizes Its RNA Substrate by Selective Engagement of Active Site Residues

Abstract

Restrictocin and related fungal endoribonucleases from the α-sarcin family site-specifically cleave the sarcin/ricin loop (SRL) on the ribosome to inhibit translation and ultimately trigger cell death. Previous studies showed that the SRL folds into a bulged-G motif and tetraloop, with restrictocin achieving a specificity of ∼1000-fold by recognizing both motifs only after the initial binding step. Here, we identify contacts within the protein−RNA interface and determine the extent to which each one contributes to enzyme specificity by examining the effect of protein mutations on the cleavage of the SRL substrate compared to a variety of other RNA substrates. As with other biomolecular interfaces, only a subset of contacts contributes to specificity. One contact of this subset is critical, with the H49A mutation resulting in quantitative loss of specificity. Maximum catalytic activity occurs when both motifs of the SRL are present, with the major contribution involving the bulged-G motif recognized by three lysine residues located adjacent to the active site: K110, K111, and K113. Our findings support a kinetic proofreading mechanism in which the active site residues H49 and, to a lesser extent, Y47 make greater catalytic contributions to SRL cleavage than to suboptimal substrates. This systematic and quantitative analysis begins to elucidate the principles governing RNA recognition by a site-specific endonuclease and may thus serve as a mechanistic model for investigating other RNA modifying enzymes.

The ribonucleolytic activity of the ribotoxin α-sarcin is not essential for in vitro protein biosynthesis inhibition

“…The possibility of H137Q and wild-type α-sarcin binding to different ribosomal locations is also plausible and the results presented do not allow discarding it. Indeed, it is known that, when first encountering the ribosomes, ribotoxins bind to different ribosomal regions to explore its electrostatic ribosomal surface and then, very quickly, locate to the SRL [28,30,31]. However, this interpretation would require a very different mechanism of protein biosynthesis inhibition for the mutant, without involving the direct participation of the SRL.…”

Chemical probing of RNA with the hydroxyl radical at single-atom resolution

“…We suggest that the backbone hydrogen bond in the GUA triple acts like the catch of a mousetrap, releasing the distorted backbone when disrupted. Since the bulged-G motif is a key recognition element for ribotoxins that cleave the SRL ( 42 ), this hydrogen bond is likely critical to the ability of ribotoxins to disable the ribosome.…”

Computational methods for predicting hotspots at protein–RNA interfaces

“…Site modification on RNA by the recognition with protein is common to many biological processes. To investigate the molecular mechanisms, the specific recognition were systematically studied on restrictocin and its substrate sarcin/ ricin loop (SRL) (Plantinga et al, 2011). To probe the contribution of specific restrictocin-SRL contacts, alanine scanning mutagenesis was performed on 11 interfacial residues (D40A, K42A, H49A, F51A, T52A, R65A, K110A, K111A, K113A, Q141A, and D143A).…”