The long-term goal of this work is to determine the principles that govern protein-RNA recognition. Interaction between protein and RNA is central to biological processes ranging from regulating gene expression to directing cell mortality. Knowledge of these principles is poorly understood, due in large part to a paucity of protein-RNA complex structures. These principles are important for understanding protein-RNA machines, such as the ribosome and the spliceosome, and protein-RNA structure and function in general. In addition, they are important for developing knowledge-based therapies against RNA-dependent infectious agents such as HIV. As a step toward the long-term goal, this proposal focuses on how the ribosome-inactivating protein restrictocin recognizes two common motifs (a tetraloop and a G-bulged cross-strand A stack) in an essential piece of ribosomal RNA. Remarkably, restrictocin, which shares 86% sequence identity with its functional homolog sarcin, cleaves only one phosphodiester bond out of the approximately 7000 found in eukaryotic ribosomal RNA. This potent and specific toxin recognizes a conserved region of ribosomal RNA called the sarcin/ricin domain (SRD) that is essential for protein synthesis. X-ray crystallographic, kinetic and energetic studies will be combined in order to obtain a deeper understanding of how restrictocin recognizes both motifs in the SRD RNA. A second series of crystallographic studies will focus on mutants in the two motifs found in the SRD RNA. These studies will provide fundamentally new insights into the principles that govern protein recognition of these two motifs, because there is an absence of related protein-RNA complex structures. Clinical interest in restrictocin and related ribosome-inactivating proteins has been invigorated by their potential use in """"""""magic bullet"""""""" therapies that direct toxins to tumor cells by linking them to tumor-specific agents such as antibodies. Determining the RNA substrate recognition surface and the contacts that are critical for restrictocin action will aid the design or selection of future ribotoxin-based therapies.
