The modified nucleoside 1-methyladenosine (m(1)A) is found at position 58 in the TPsiC loop of many eukaryotic tRNAs. The absence of m(1)A from all tRNAs in Saccharomyces cerevisiae mutants lacking Gcd10p elicits severe defects in processing and stability of initiator methionine tRNA (tRNA(i)(Met)). Gcd10p is found in a complex with Gcd14p, which contains conserved motifs for binding S-adenosylmethionine (AdoMet). These facts, plus our demonstration that gcd14Delta cells lacked m(1)A, strongly suggested that Gcd10p/Gcd14p complex is the yeast tRNA(m(1)A)methyltransferase [(m(1)A)MTase]. Supporting this prediction, affinity-purified Gcd10p/Gcd14p complexes used AdoMet as a methyl donor to synthesize m(1)A in either total tRNA or purified tRNA(i)(Met) lacking only this modification. Kinetic analysis of the purified complex revealed K(M) values for AdoMet or tRNA(i)(Met) of 5.0 microM and 2.5 nM, respectively. Mutations in the predicted AdoMet-binding domain destroyed GCD14 function in vivo and (m(1)A)MTase activity in vitro. Purified Flag-tagged Gcd14p alone had no enzymatic activity and was severely impaired for tRNA-binding compared with the wild-type complex, suggesting that Gcd10p is required for tight binding of the tRNA substrate. Our results provide a demonstration of a two-component tRNA MTase and suggest that binding of AdoMet and tRNA substrates depends on different subunits of the complex.
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| Gene/Complex | Qualifier | Gene Ontology Term | Annotation Extension | Evidence | Source | Assigned On |
|---|---|---|---|---|---|---|
| GCD14 | part of | tRNA (m1A) methyltransferase complex | IDA | SGD | 2013-08-07 | |
| GCD14 | contributes to | tRNA (adenine(58)-N1)-methyltransferase activity | IMP | SGD | 2023-10-12 | |
| GCD14 | contributes to | tRNA (adenine(58)-N1)-methyltransferase activity | IDA | SGD | 2023-10-12 | |
| GCD14 | part of | tRNA (m1A) methyltransferase complex | IPI with GCD10 | SGD | 2013-08-07 | |
| GCD14 | involved in | tRNA methylation | IDA | SGD | 2013-08-07 | |
| GCD10 | involved in | tRNA methylation | IDA | SGD | 2013-08-07 | |
| GCD10 | part of | tRNA (m1A) methyltransferase complex | IPI with GCD14 | SGD | 2013-08-07 | |
| GCD10 | contributes to | tRNA (adenine(58)-N1)-methyltransferase activity | IDA | SGD | 2023-10-12 | |
| GCD10 | part of | tRNA (m1A) methyltransferase complex | IDA | SGD | 2013-08-07 |
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| Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Increase the total number of rows showing on this page by using the pull-down located below the table, or use the page scroll at the table's top right to browse through the table's pages; use the arrows to the right of a column header to sort by that column; filter the table using the "Filter" box at the top of the table; click on the small "i" buttons located within a cell for an annotation to view further details about experiment type and any other genes involved in the interaction.