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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Biophysical Chemistry Study Section (BBCB)
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Lewis, Catherine D
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University of Chicago
Schools of Medicine
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Korennykh, Alexei V; Plantinga, Matthew J; Correll, Carl C et al. (2007) Linkage between substrate recognition and catalysis during cleavage of sarcin/ricin loop RNA by restrictocin. Biochemistry 46:12744-56
Korennykh, Alexei V; Correll, Carl C; Piccirilli, Joseph A (2007) Evidence for the importance of electrostatics in the function of two distinct families of ribosome inactivating toxins. RNA 13:1391-6
Korennykh, Alexei V; Piccirilli, Joseph A; Correll, Carl C (2006) The electrostatic character of the ribosomal surface enables extraordinarily rapid target location by ribotoxins. Nat Struct Mol Biol 13:436-43
Chan, Yuen-Ling; Correll, Carl C; Wool, Ira G (2004) The location and the significance of a cross-link between the sarcin/ricin domain of ribosomal RNA and the elongation factor-G. J Mol Biol 337:263-72
Gerczei, Timea; Correll, Carl C (2004) Imp3p and Imp4p mediate formation of essential U3-precursor rRNA (pre-rRNA) duplexes, possibly to recruit the small subunit processome to the pre-rRNA. Proc Natl Acad Sci U S A 101:15301-6
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Correll, Carl C; Swinger, Kerren (2003) Common and distinctive features of GNRA tetraloops based on a GUAA tetraloop structure at 1.4 A resolution. RNA 9:355-63
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