Use of nucleotide analogs by class I and class II CCA-adding enzymes (tRNA nucleotidyltransferase): deciphering the basis for nucleotide selection

We explored the specificity and nature of the nucleotide-binding pocket of the CCA-adding enzyme (tRNA nucleotidyltransferase) by using CTP and ATP analogs as substrates for a panel of class I and class II enzymes. Overall, class I and class II enzymes displayed remarkably similar substrate requirements, implying that the mechanism of CCA addition is conserved between enzyme classes despite the absence of obvious sequence homology outside the active site signature sequence. CTP substrates are more tolerant of base modifications than ATP substrates, but sugar modifications prevent incorporation of both CTP and ATP analogs by class I and class II enzymes. Use of CTP analogs (zebularine, pseudoisocytidine, 6-azacytidine, but not 6-azauridine) suggests that base modifications generally do not interfere with recognition or incorporation of CTP analogs by either class I or class II enzymes, and that UTP is excluded because N-3 is a positive determinant and/or O-4 is an antideterminant. Use of ATP analogs (N6-methyladenosine, diaminopurine, purine, 2-aminopurine, and 7-deaza-adenosine, but not guanosine, deoxyadenosine, 2'-O-methyladenosine, 2'-deoxy-2'-fluoroadenosine, or inosine) suggests that base modifications generally do not interfere with recognition or incorporation of ATP analogs by either class I or class II enzymes, and that GTP is excluded because N-1 is a positive determinant and/or the 2-amino and 6-keto groups are antideterminants. We also found that the 3'-terminal sequence of the growing tRNA substrate can affect the efficiency or specificity of subsequent nucleotide addition. Our data set should allow rigorous evaluation of structural hypotheses for nucleotide selection based on existing and future crystal structures.