Minimal requirements for reverse polymerization and tRNA repair by tRNA<sup>His</sup> guanylyltransferase
Bibliographic record
Abstract
tRNAHis guanylyltransferase (Thg1) has unique reverse (3’-5’) polymerase activity occurring in all three domains of life. Most eukaryotic Thg1 homologs are essential genes involved in tRNAHis maturation. These enzymes normally catalyze a single 5’ guanylation of tRNAHis lacking the essential G−1 identity element required for aminoacylation. Recent studies suggest that archaeal type Thg1, which includes most archaeal and bacterial Thg1 enzymes is phylogenetically distant from eukaryotic Thg1. Thg1 is evolutionarily related to canonical 5’-3’ forward polymerases but catalyzes reverse 3’-5’polymerization. Similar to its forward polymerase counterparts, Thg1 encodes the conserved catalytic palm domain and fingers domain. Here we investigate the minimal requirements for reverse polymerization. We show that the naturally occurring minimal Thg1 enzyme from Ignicoccus hospitalis (IhThg1), which lacks parts of the conserved fingers domain, is catalytically active. And adds all four natural nucleotides to RNA substrates, we further show that the entire fingers domain of Methanosarcina acetivorans Thg1 and Pyrobaculum aerophilum Thg1 (PaThg1) is dispensable for enzymatic activity. In addition, we identified residues in yeast Thg1 that play a part in preventing extended polymerization. Mutation of these residues with alanine resulted in extended reverse polymerization. PaThg1 was found to catalyze extended, template dependent tRNA repair, adding up to 13 nucleotides to a truncated tRNAHis substrate. Sequencing results suggest that PaThg1 fully restored the near correct sequence of the D- and acceptor stem, but also produced incompletely and incorrectly repaired tRNA products. This research forms the basis for future engineering efforts towards a high fidelity, template dependent reverse polymerase.
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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 | 0.001 |
| 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 | 0.001 | 0.000 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
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".