Two-step mechanism and step-arrest mutants of <i>Runella slithyformis</i> NAD<sup>+</sup>-dependent tRNA 2′-phosphotransferase Tpt1
Bibliographic record
Abstract
Tpt1 catalyzes the transfer of an internal 2′-monophosphate moiety (2′-PO 4 ) from a “branched” 2′-PO 4 RNA splice junction to NAD + to form a “clean” 2′-OH, 3′-5′ phosphodiester junction, ADP-ribose 1″-2″ cyclic phosphate, and nicotinamide. First discovered as an essential component of the Saccharomyces cerevisiae tRNA splicing machinery, Tpt1 is widely distributed in nature, including in taxa that have no yeast-like RNA splicing system. Here we characterize the RslTpt1 protein from the bacterium Runella slithyformis , in which Tpt1 is encoded within a putative RNA repair gene cluster. We find that (i) expression of RslTpt1 in yeast complements a lethal tpt1 Δ knockout, and (ii) purified recombinant RslTpt1 is a bona fide NAD + -dependent 2′-phosphotransferase capable of completely removing an internal 2′-phosphate from synthetic RNAs. The in vivo activity of RslTpt1 is abolished by alanine substitutions for conserved amino acids Arg16, His17, Arg64, and Arg119. The R64A, R119A, and H17A mutants accumulate high levels of a 2′-phospho-ADP-ribosylated RNA reaction intermediate (2′-P-ADPR, evanescent in the wild-type RslTpt1 reaction), which is converted slowly to a 2′-OH RNA product. The R16A mutant is 300-fold slower than wild-type RslTpt1 in forming the 2′-P-ADPR intermediate. Whereas wild-type RsTpt1 rapidly converts the isolated 2′-P-ADPR intermediate to 2′-OH product in the absence of NAD + , the H17A, R119A, R64A, and R16A mutant are slower by factors of 3, 33, 210, and 710, respectively. Our results identify active site constituents involved in the catalysis of step 1 and step 2 of the Tpt1 reaction pathway.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| 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) | 0.000 | 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 teacher head, 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".