The main goal of the project is to understand how specific RNA structures contribute to RNA catalysis. The major experimental system will continue to be the self-splicing IVS (intervening sequence) of Tetrahymena and ribozymes (RNA enzymes) derived from the IVS. Additional goals are to develop a set of sequence- specific RNA cleavage enzymes that may be useful as tools for RNA molecular biology and to explore the generality of RNA catalysis in new directions.
Specific aims are the following: (1) Obtain a 3-dimensional view of the active site of the Tetrahymena IVS RNA with and without the substrates bound. Approaches will include chemical modification, cleavage of the RNA by an active-site-directed inhibitor, and UV crosslinking. (2) Identify nucleotides involved in substrate- binding and catalysis using site-specific and random mutagenesis. A particular model of the 3-dimensional structure of the catalytic core will be tested. (3) Further explore the activity of the ribozyme as a sequence-specific endoribonuclease. Twenty active- site variants with altered substrate specificity will be characterized. (4) Test the idea that nuclear mRNA splicing is at least in part catalyzed by small nuclear RNAs using the Saccharomyces cerevisiae system. (5) Test the idea that mRNA stability might in some cases be determined by the presence of self-cleavage sites. The Tetrahymena IVS RNA provides an unusually amenable system for learning about structure-function relationships in RNA and biological catalysis in general. It is likely that many of he findings will be applicable to other systems where RNA is in catalysis, including other RNA processing reactions and protein synthesis.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
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University of Colorado at Boulder
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Nandakumar, Jayakrishnan; Cech, Thomas R (2012) DNA-induced dimerization of the single-stranded DNA binding telomeric protein Pot1 from Schizosaccharomyces pombe. Nucleic Acids Res 40:235-44
Taylor, Derek J; Podell, Elaine R; Taatjes, Dylan J et al. (2011) Multiple POT1-TPP1 proteins coat and compact long telomeric single-stranded DNA. J Mol Biol 410:10-7
Zappulla, David C; Goodrich, Karen J; Arthur, Julian R et al. (2011) Ku can contribute to telomere lengthening in yeast at multiple positions in the telomerase RNP. RNA 17:298-311
Latrick, Chrysa M; Cech, Thomas R (2010) POT1-TPP1 enhances telomerase processivity by slowing primer dissociation and aiding translocation. EMBO J 29:924-33
Zappulla, David C; Roberts, Jennifer N; Goodrich, Karen J et al. (2009) Inhibition of yeast telomerase action by the telomeric ssDNA-binding protein, Cdc13p. Nucleic Acids Res 37:354-67
Subramanian, Lakxmi; Moser, Bettina A; Nakamura, Toru M (2008) Recombination-based telomere maintenance is dependent on Tel1-MRN and Rap1 and inhibited by telomerase, Taz1, and Ku in fission yeast. Mol Cell Biol 28:1443-55
Mandell, Jeffrey G; Goodrich, Karen J; Bahler, Jurg et al. (2005) Expression of a RecQ helicase homolog affects progression through crisis in fission yeast lacking telomerase. J Biol Chem 280:5249-57
Zappulla, David C; Goodrich, Karen; Cech, Thomas R (2005) A miniature yeast telomerase RNA functions in vivo and reconstitutes activity in vitro. Nat Struct Mol Biol 12:1072-7
Zappulla, David C; Cech, Thomas R (2004) Yeast telomerase RNA: a flexible scaffold for protein subunits. Proc Natl Acad Sci U S A 101:10024-9
Aigner, Stefan; Cech, Thomas R (2004) The Euplotes telomerase subunit p43 stimulates enzymatic activity and processivity in vitro. RNA 10:1108-18

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