The HD Domain of the Escherichia coli tRNA Nucleotidyltransferase Has 2′,3′-Cyclic Phosphodiesterase, 2′-Nucleotidase, and Phosphatase Activities
Bibliographic record
Abstract
In all mature tRNAs, the 3'-terminal CCA sequence is synthesized or repaired by a template-independent nucleotidyltransferase (ATP(CTP):tRNA nucleotidyltransferase; EC 2.7.7.25). The Escherichia coli enzyme comprises two domains: an N-terminal domain containing the nucleotidyltransferase activity and an uncharacterized C-terminal HD domain. The HD motif defines a superfamily of metal-dependent phosphohydrolases that includes a variety of uncharacterized proteins and domains associated with nucleotidyltransferases and helicases from bacteria, archaea, and eukaryotes. The C-terminal HD domain in E. coli tRNA nucleotidyltransferase demonstrated Ni(2+)-dependent phosphatase activity toward pyrophosphate, canonical 5'-nucleoside tri- and diphosphates, NADP, and 2'-AMP. Assays with phosphodiesterase substrates revealed surprising metal-independent phosphodiesterase activity toward 2',3'-cAMP, -cGMP, and -cCMP. Without metal or in the presence of Mg(2+), the tRNA nucleotidyltransferase hydrolyzed 2',3'-cyclic substrates with the formation of 2'-nucleotides, whereas in the presence of Ni(2+), the protein also produced some 3'-nucleotides. Mutations at the conserved His-255 and Asp-256 residues comprising the C-terminal HD domain of this protein inactivated both phosphodiesterase and phosphatase activities, indicating that these activities are associated with the HD domain. Low concentrations of the E. coli tRNA (10 nm) had a strong inhibiting effect on both phosphatase and phosphodiesterase activities. The competitive character of inhibition by tRNA suggests that it might be a natural substrate for these activities. This inhibition was completely abolished by the addition of Mg(2+), Mn(2+), or Ca(2+), but not Ni(2+). The data suggest that the phosphohydrolase activities of the HD domain of the E. coli tRNA nucleotidyltransferase are involved in the repair of the 3'-CCA end of tRNA.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".