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. 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 somehow serve as a template for nucleotide addition. Although the two nonhomologous classes of CCA adding enzymes share only a conserved nucleotidyltransferase motif, both classes have a single active site, bind primarily to the top half (""""""""minihelix"""""""") of tRNA, and do not translocate along the tRNA during CCA addition. To explain how three nucleotides can be added without movement of the tRNA or active site, we proposed that the growing 3' terminus of the tRNA would progressively scrunch into a pocket, allowing the solitary active site to reuse a single nucleotide-binding site. How the folded 3' terminus would determine the specificity of CTP or ATP addition was not clear, but nucleotide addition would cease when the scrunching pocket was full. To explore this model, we now propose a thorough mutational analysis of the active site, scrunching pocket, and tRNA-binding regions of four different enzymes: the archaeal class I Sulfolobus shibatae CCA-adding enzyme (Aim 1), the eubacterial class II Bacillus stearothermophilus CCA-adding enzyme (Aim 2), and the unusual eubacterial class II CC- and A-adding enzymes of Aquifex aeolicus (Aim 3). In addition, we will mutate the dimerization interfaces of both class I and class II enzymes to determine whether the functional unit of these enzymes is a monomer or multimer (Aim 4); we will obtain crystal or cocrystal structures of selected mutants characterized in the previous four aims (Aim 5); and, as the ultimate test of our understanding, we will use structure-based protein redesign of the nucleotide binding site and scrunching pocket to create mutants with altered sequence specificity (Aim 6). Our experiments should reveal the detailed mechanism of the only enzyme that templates specific nucleotide sequences using protein instead of nucleic acid; shed light on the generality of the scrunching mechanisms used by many polymerases to facilitate initiation as well as editing of misincorporated nucleotides at the growing 3' terminus; and possibly explain why this ancient essential activity is performed today by two highly divergent protein scaffolds (class I and II).

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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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