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1.
J Biol Chem ; 299(9): 105149, 2023 09.
Article in English | MEDLINE | ID: mdl-37567477

ABSTRACT

Alanyl-tRNA synthetase retains a conserved prototype structure throughout its biology. Nevertheless, its C-terminal domain (C-Ala) is highly diverged and has been shown to play a role in either tRNA or DNA binding. Interestingly, we discovered that Caenorhabditis elegans cytoplasmic C-Ala (Ce-C-Alac) robustly binds both ligands. How Ce-C-Alac targets its cognate tRNA and whether a similar feature is conserved in its mitochondrial counterpart remain elusive. We show that the N- and C-terminal subdomains of Ce-C-Alac are responsible for DNA and tRNA binding, respectively. Ce-C-Alac specifically recognized the conserved invariant base G18 in the D-loop of tRNAAla through a highly conserved lysine residue, K934. Despite bearing little resemblance to other C-Ala domains, C. elegans mitochondrial C-Ala robustly bound both tRNAAla and DNA and maintained targeting specificity for the D-loop of its cognate tRNA. This study uncovers the underlying mechanism of how C. elegans C-Ala specifically targets the D-loop of tRNAAla.


Subject(s)
Alanine-tRNA Ligase , Caenorhabditis elegans , Nucleotide Motifs , RNA, Transfer, Ala , Animals , Alanine-tRNA Ligase/chemistry , Alanine-tRNA Ligase/metabolism , Caenorhabditis elegans/enzymology , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Conserved Sequence , Cytoplasm/enzymology , DNA/chemistry , DNA/metabolism , Ligands , Lysine/metabolism , Mitochondria/enzymology , Protein Domains , RNA, Transfer, Ala/chemistry , RNA, Transfer, Ala/metabolism , Substrate Specificity , Nucleic Acid Conformation
2.
Nucleic Acids Res ; 50(17): 10015-10025, 2022 09 23.
Article in English | MEDLINE | ID: mdl-36107775

ABSTRACT

tRNAHis guanylyltransferase (Thg1) catalyzes the 3'-5' incorporation of guanosine into position -1 (G-1) of tRNAHis. G-1 is unique to tRNAHis and is crucial for recognition by histidyl-tRNA synthetase (HisRS). Yeast Thg1 requires ATP for G-1 addition to tRNAHis opposite A73, whereas archaeal Thg1 requires either ATP or GTP for G-1 addition to tRNAHis opposite C73. Paradoxically, human Thg1 (HsThg1) can add G-1 to tRNAsHis with A73 (cytoplasmic) and C73 (mitochondrial). As N73 is immediately followed by a CCA end (positions 74-76), how HsThg1 prevents successive 3'-5' incorporation of G-1/G-2/G-3 into mitochondrial tRNAHis (tRNAmHis) through a template-dependent mechanism remains a puzzle. We showed herein that mature native human tRNAmHis indeed contains only G-1. ATP was absolutely required for G-1 addition to tRNAmHis by HsThg1. Although HsThg1 could incorporate more than one GTP into tRNAmHisin vitro, a single-GTP incorporation prevailed when the relative GTP level was low. Surprisingly, HsThg1 possessed a tRNA-inducible GTPase activity, which could be inhibited by ATP. Similar activity was found in other high-eukaryotic dual-functional Thg1 enzymes, but not in yeast Thg1. This study suggests that HsThg1 may downregulate the level of GTP through its GTPase activity to prevent multiple-GTP incorporation into tRNAmHis.


Subject(s)
Nucleotidyltransferases/metabolism , RNA, Transfer, His , Adenosine Triphosphate , GTP Phosphohydrolases/genetics , Guanosine , Guanosine Triphosphate/metabolism , Histidine-tRNA Ligase , Humans , RNA, Transfer , RNA, Transfer, His/genetics , RNA, Transfer, His/metabolism , Saccharomyces cerevisiae/metabolism
3.
Ecotoxicol Environ Saf ; 172: 432-438, 2019 May 15.
Article in English | MEDLINE | ID: mdl-30735975

ABSTRACT

Diuron is an herbicide, which is used to control a wide variety of annual and perennial broadleaf, grassy weeds, and mosses. However, the toxicity of diuron in HepG2 cells and zebrafish embryos was unclear. In this study, HpeG2 cells and zebrafish embryos were exposed to different concentrations of diuron for 24 h and 48 h, respectively. Results reveal the diuron caused cytotoxicity and the generation of reactive oxygen species (ROS) in the treated HepG2 cells. The effects of diuron on the expression of catalase and superoxide dismutase (SOD1 and SOD2), an antioxidant enzyme, were investigated. Results showed that only SOD1 was significantly induced after treated diuron 48 h, but the expression of catalase and SOD2 was unaffected. Additionally, the cytotoxicity of diuron was not attenuated in cells pretreated with of N-acetyl-cysteine (NAC), a well-known antioxidant, indicating that oxidative stress could not contribute to cellular death in the treated HepG2 cells. In zebrafish embryos, results from proteomic analysis show that 332 differentially upregulated proteins and 199 down-regulated proteins were detected in the treated embryos (P < 0.05). In addition to the up-regulated antioxidant proteins (prdx3, cat, prdx4, txnrd1, prdx1, sod1, prdx2, and sod2), some decreased proteins were related to cytoskeleton formation, tight junction, and gap junction, which could be related to the malformation of the treated zebrafish embryos. In summary, diuron caused cytotoxicity in HepG2 cells, and the mechanisms of toxicity in zebrafish were addressed using the proteomic analysis.


Subject(s)
Diuron/toxicity , Embryo, Nonmammalian/drug effects , Herbicides/toxicity , Animals , Catalase/metabolism , Hep G2 Cells , Humans , Oxidative Stress , Proteomics , Reactive Oxygen Species/metabolism , Superoxide Dismutase/metabolism , Toxicity Tests , Zebrafish
4.
PLoS One ; 9(4): e94659, 2014.
Article in English | MEDLINE | ID: mdl-24743154

ABSTRACT

Two oligomeric types of glycyl-tRNA synthetase (GlyRS) are found in nature: a α2 type and a α2ß2 type. The former has been identified in all three kingdoms of life and often pairs with tRNAGly that carries an A73 discriminator base, while the latter is found only in bacteria and chloroplasts and is almost always coupled with tRNAGly that contains U73. In the yeast Saccharomyces cerevisiae, a single GlyRS gene, GRS1, provides both the cytoplasmic and mitochondrial functions, and tRNAGly isoacceptors in both compartments possess A73. We showed herein that Homo sapiens and Arabidopsis thaliana cytoplasmic GlyRSs (both α2-type enzymes) can rescue both the cytoplasmic and mitochondrial defects of a yeast grs1- strain, while Escherichia coli GlyRS (a α2ß2-type enzyme) and A. thaliana organellar GlyRS (a (αß)2-type enzyme) failed to rescue either defect of the yeast mull allele. However, a head-to-tail αß fusion of E. coli GlyRS effectively supported the mitochondrial function. Our study suggests that a α2-type eukaryotic GlyRS may be functionally substituted with a α2ß2-type bacterial cognate enzyme despite their remote evolutionary relationships.


Subject(s)
Eukaryota/enzymology , Evolution, Molecular , Glycine-tRNA Ligase/metabolism , Aminoacylation , Animals , Bacteria/enzymology , Base Sequence , Cloning, Molecular , Gene Knockout Techniques , Glycine-tRNA Ligase/chemistry , Glycine-tRNA Ligase/deficiency , Glycine-tRNA Ligase/genetics , Humans , Mitochondria/genetics , Mitochondria/metabolism , Protein Multimerization , Protein Structure, Quaternary , Protein Transport
5.
BMC Microbiol ; 10: 188, 2010 Jul 09.
Article in English | MEDLINE | ID: mdl-20618922

ABSTRACT

BACKGROUND: Previous studies in Saccharomyces cerevisiae showed that ALA1 (encoding alanyl-tRNA synthetase) and GRS1 (encoding glycyl-tRNA synthetase) respectively use ACG and TTG as their alternative translation initiator codons. To explore if any other non-ATG triplets can act as initiator codons in yeast, ALA1 was used as a reporter for screening. RESULTS: We show herein that except for AAG and AGG, all triplets that differ from ATG by a single nucleotide were able to serve as initiator codons in ALA1. Among these initiator codons, TTG, CTG, ACG, and ATT had ~50% initiating activities relative to that of ATG, while GTG, ATA, and ATC had ~20% initiating activities relative to that of ATG. Unexpectedly, these non-AUG initiator codons exhibited different preferences toward various sequence contexts. In particular, GTG was one of the most efficient non-ATG initiator codons, while ATA was essentially inactive in the context of GRS1. CONCLUSION: This finding indicates that a sequence context that is favorable for a given non-ATG initiator codon might not be as favorable for another.


Subject(s)
Alanine-tRNA Ligase/metabolism , Codon, Initiator , Peptide Chain Initiation, Translational , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Alanine-tRNA Ligase/genetics , Amino Acid Sequence , Base Sequence , Molecular Sequence Data , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics
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