Dataset Information

Functional and structural investigation of N-terminal domain of the SpTad2/3 heterodimeric tRNA deaminase.

ABSTRACT: Editing is a post-transcriptional process that changes the content of nucleic acids occurring on both DNA and RNA levels. Inosine at position 34 in tRNA is one such example, commonly produced via the deamination of A34, catalyzed by adenosine deaminase acting on tRNA (ADAT or Tad). The formation of inosine is essential for cell viability. The eukaryotic deaminases normally consist of the catalytic subunit Tad2 and the structural subunit Tad3, but the catalytic process is poorly understood. Despite the conservation of the (pseudo-) catalytic domains, the heterodimeric enzyme Tad2/3 also possesses additional domains that could exhibit novel functions. Here we present the structure of the N-terminal domain of the Schizosaccharomyces pombe Tad2/3 heterodimeric tRNA(A34) deaminase (N-SpTad2), which shares ~30% sequence identities with uridine-cytidine or pantothenate kinases, but lacks the predicted kinase functions. While biochemical assays indicated that the domain is not a nucleic-acid binder, it is able to significantly influence the A34-tRNA deamination activity of the holoenzyme. Through co-expression and purification analyses, we deduce that N-SpTad2 plays a role in mediating protein-protein contacts and enhancing the stability and solubility of SpTad2/3, without which the deaminase is not functional. Taken together, our structural and biochemical studies highlighted the importance of the additional domains to the intrinsic deaminase functions of heterodimeric Tad2/3 enzymes and promoted our understanding on this essential post-transcriptional tRNA modification.

SUBMITTER: Liu X

PROVIDER: S-EPMC8217354 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:The cystic fibrosis transmembrane conductance regulator (CFTR), encoded by the gene mutated in cystic fibrosis patients, belongs to the family of ATP-binding cassette (ABC) proteins, but, unlike other members, functions as a chloride channel. CFTR is activated by protein kinase A (PKA)-mediated phosphorylation of multiple sites in its regulatory domain, and gated by binding and hydrolysis of ATP at its two nucleotide binding domains (NBD1, NBD2). The recent crystal structure of NBD1 from mouse CFTR (Lewis, H.A., S.G. Buchanan, S.K. Burley, K. Conners, M. Dickey, M. Dorwart, R. Fowler, X. Gao, W.B. Guggino, W.A. Hendrickson, et al. 2004. EMBO J. 23:282-293) identified two regions absent from structures of all other NBDs determined so far, a "regulatory insertion" (residues 404-435) and a "regulatory extension" (residues 639-670), both positioned to impede formation of the putative NBD1-NBD2 dimer anticipated to occur during channel gating; as both segments appeared highly mobile and both contained consensus PKA sites (serine 422, and serines 660 and 670, respectively), it was suggested that their phosphorylation-linked conformational changes might underlie CFTR channel regulation. To test that suggestion, we coexpressed in Xenopus oocytes CFTR residues 1-414 with residues 433-1480, or residues 1-633 with 668-1480, to yield split CFTR channels (called 414+433 and 633+668) that lack most of the insertion, or extension, respectively. In excised patches, regulation of the resulting CFTR channels by PKA and by ATP was largely normal. Both 414+433 channels and 633+668 channels, as well as 633(S422A)+668 channels (lacking both the extension and the sole PKA consensus site in the insertion), were all shut during exposure to MgATP before addition of PKA, but activated like wild type (WT) upon phosphorylation; this indicates that inhibitory regulation of nonphosphorylated WT channels depends upon neither segment. Detailed kinetic analysis of 414+433 channels revealed intact ATP dependence of single-channel gating kinetics, but slightly shortened open bursts and faster closing from the locked-open state (elicited by ATP plus pyrophosphate or ATP plus AMPPNP). In contrast, 633+668 channel function was indistinguishable from WT at both macroscopic and microscopic levels. We conclude that neither nonconserved segment is an essential element of PKA- or nucleotide-dependent regulation.

Dataset Information

Functional and structural investigation of N-terminal domain of the SpTad2/3 heterodimeric tRNA deaminase.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets