Kinetic and structural insights into the requirement of fungal tRNA ligase for a 2′-phosphate end Journal Article
| Authors: | Ghosh, S.; Shuman, S. |
| Article Title: | Kinetic and structural insights into the requirement of fungal tRNA ligase for a 2′-phosphate end |
| Abstract: | Fungal RNA ligase (LIG) is an essential tRNA splicing enzyme that joins 3′′-OH,2′′-PO4 and 5′′-PO4 RNA ends to form a 2′′-PO4,3′′-5′′ phosphodiester splice junction. Sealing entails three divalent cation-dependent adenylate transfer steps. First, LIG reacts with ATP to form a covalent ligase-(lysyl-Nζ)-AMP intermediate and displace pyrophosphate. Second, LIG transfers AMP to the 5′′-PO4 RNA terminus to form an RNA-adenylate intermediate (A5′′pp5′′RNA). Third, LIG directs the attack of an RNA 3′′-OH on AppRNA to form the splice junction and displace AMP. A defining feature of fungal LIG vis-à-vis canonical polynucleotide ligases is the requirement for a 2′′-PO4 to synthesize a 3′′–5′′ phosphodiester bond. Fungal LIG consists of an N-terminal adenylyltransferase domain and a unique C-terminal domain. The C-domain of Chaetomium thermophilum LIG (CthLIG) engages a sulfate anion thought to be a mimetic of the terminal 2′′-PO4. Here, we interrogated the contributions of the C-domain and the conserved sulfate ligands (His227, Arg334, Arg337) to ligation of a pRNA2′′p substrate. We find that the C-domain is essential for end-joining but dispensable for ligase adenylylation. Mutations H227A, R334A, and R337A slowed the rate of step 2 RNA adenylation by 420-fold, 120-fold, and 60-fold, respectively, vis-à-vis wild-type CthLIG. An R334A-R337A double-mutation slowed step 2 by 580-fold. These results fortify the case for the strictly conserved His–Arg–Arg triad as the enforcer of the 2′′-PO4 end-specificity of fungal tRNA ligases and as a target for small molecule interdiction of fungal tRNA splicing. © 2024 Ghosh and Shuman. |
| Keywords: | genetics; metabolism; enzymology; chemistry; kinetics; substrate specificity; models, molecular; enzyme specificity; phosphate; phosphates; trna splicing; rna ligase (atp); fungal protein; molecular model; rna splicing; rna, fungal; adenosine phosphate; rna repair; fungal proteins; adenosine monophosphate; rna ligase; fungal rna; rna end recognition; chaetomium; chaetomium thermophilum |
| Journal Title: | RNA |
| Volume: | 30 |
| Issue: | 10 |
| ISSN: | 1355-8382 |
| Publisher: | Cold Spring Harbor Laboratory Press |
| Date Published: | 2024-10-01 |
| Start Page: | 1306 |
| End Page: | 1314 |
| Language: | English |
| DOI: | 10.1261/rna.080120.124 |
| PUBMED: | 39013577 |
| PROVIDER: | scopus |
| PMCID: | PMC11404444 |
| DOI/URL: | |
| Notes: | Article -- Source: Scopus |
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