The CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase] builds and repairs the 3' terminal CCA sequence of all tRNAs by adding one nucleotide at a time, using CTP and ATP as substrates. Unlike all other sequence-specific RNA and DNA polymerases, the CCA-adding enzyme does not use a nucleic acid template. Thus the protein itself must serve as a template, or the enzyme must use a novel mechanism to specify nucleotide addition. We have shown that the CCA-adding enzyme has only a single active site, that the enzyme binds primarily to the acceptor stem (""""""""top half"""""""") of tRNA, and that the tRNA remains immobile on the enzyme surface during addition of CCA. To explain how three nucleotides can be added to tRNA without movement of either the tRNA or the active site, we proposed that the growing 3' terminus of the tRNA progressively refolds to allow the solitary active site to reuse a single nucleotide binding site. The ATP binding site would be created collaboratively by the refolded CC terminus and the enzyme, and nucleotide addition would cease when the nucleotide binding pocket is full. The template for CCA addition would therefore be a dynamic ribonucleoprotein structure, in a mechanism we call collaborative templating. Here we propose to study the CCA-adding enzyme in biochemical detail. The experiments will test the collaborative templating model, and provide a wealth of new information about the CCA-adding enzyme. Specifically, we will use photochemical crosslinking and hydroxyl radical footprinting to identify amino acid residues in the immediate vicinity of the active site, the nucleotide binding pocket, and the tRNA binding site; we will ask whether mutations in these residues change the specificity of CCA addition as predicted by the model; we will use nucleotide analogues to define the nature of the nucleotide binding pocket; and we will continue our efforts to crystallize or cocrystallize the CCA- adding enzyme with tRNA substrates. In principle, cocrystal structures of the enzyme with the three substrates (tRNA-N, tRNA- NC, tRNA-NCC) and the mature tRNA product (tRNA-NCCA where N is the """"""""discriminator base"""""""") would provide a moving picture of this unusual enzyme in action.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Biochemistry Study Section (BIO)
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Ikeda, Richard A
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University of Washington
Schools of Medicine
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Cho, Hyundae D; Sood, Vanita D; Baker, David et al. (2008) On the role of a conserved, potentially helix-breaking residue in the tRNA-binding alpha-helix of archaeal CCA-adding enzymes. RNA 14:1284-9
Cho, Hyundae D; Verlinde, Christophe L M J; Weiner, Alan M (2007) Reengineering CCA-adding enzymes to function as (U,G)- or dCdCdA-adding enzymes or poly(C,A) and poly(U,G) polymerases. Proc Natl Acad Sci U S A 104:54-9
Cho, Hyundae D; Chen, Yu; Varani, Gabriele et al. (2006) A model for C74 addition by CCA-adding enzymes: C74 addition, like C75 and A76 addition, does not involve tRNA translocation. J Biol Chem 281:9801-11
Cho, Hyundae D; Verlinde, Christophe L; Weiner, Alan M (2005) Archaeal CCA-adding enzymes: central role of a highly conserved beta-turn motif in RNA polymerization without translocation. J Biol Chem 280:9555-66
Cho, HyunDae D; Weiner, Alan M (2004) A single catalytically active subunit in the multimeric Sulfolobus shibatae CCA-adding enzyme can carry out all three steps of CCA addition. J Biol Chem 279:40130-6
Xiong, Yong; Li, Fang; Wang, Jimin et al. (2003) Crystal structures of an archaeal class I CCA-adding enzyme and its nucleotide complexes. Mol Cell 12:1165-72
Tomita, Kozo; Weiner, Alan M (2002) Closely related CC- and A-adding enzymes collaborate to construct and repair the 3'-terminal CCA of tRNA in Synechocystis sp. and Deinococcus radiodurans. J Biol Chem 277:48192-8
Li, Fang; Xiong, Yong; Wang, Jimin et al. (2002) Crystal structures of the Bacillus stearothermophilus CCA-adding enzyme and its complexes with ATP or CTP. Cell 111:815-24
Tomita, K; Weiner, A M (2001) Collaboration between CC- and A-adding enzymes to build and repair the 3'-terminal CCA of tRNA in Aquifex aeolicus. Science 294:1334-6