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).