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From: Bagus Priambodo

To: Titi Rindi Antika

An aminoacyl tRNA synthetase (aaRS) is an enzyme that attaches the appropriate amino
acid onto its tRNA. It does so by catalyzing the esterification of a specific cognate amino
acid or its precursor to one of all its compatible cognate tRNAs to form an aminoacyl-
tRNA. Protein translation was occured in cytoplasm and mitochondria, also using different
aaRSs respectively. for example, Glycyl-tRNA synthetase (GlyRS) was encoded by the same
nuclear gene through alternative initiation of translation. There is only one GlyRS gene in
the nuclear genome of Caenorhabditis elegans, namely, CeGRS1. CeGlyRS acquired two
functional domains, a mitochondrial appended domain (MAD) and a WHEP domain, during
evolution.Because mitochondrial tRNAs in this organism typically possess a defective TC
hairpin, the aim of this study is to evaluate how enzyme(s) encoded by this gene can
efficiently charge such a tRNA species. For the methods firstly, Plasmids construction was
performed using Real Time PCR and gene-specific primers. The PCR-amplified fragment
was digested with EagI and NdeI and then cloned into the EagI/NdeI sites of pADH.
Complementation assay for cytoplasmic and mitochondrial activity using yeast GRS1 and
knock-out with a test plasmid which carried a LEU2 marker. Then the cytoplasmic and
mitochondria phenotypes were tested on 5-FOA and YPG respectively. Those yeast cells
were observed with fluorencence microscopy using specific stain. several following
treatment would checked using gelm electrophoresis and Western blotting. Results shows,
CeGRS1 suggested that this gene is dual functional, with ATG1 and ATG65 being the
respective initiator codons of the mitochondrial and cytoplasmic forms of CeGlyRS, then
they present evidence herein that cytoplasmic and mitochondrial forms of Caenorhabditis
elegans glycyl-tRNA synthetase (CeGlyRS) are encoded by the same gene (CeGRS1)
through alternative initiation of translation. The cytoplasmic form possessed an N-terminal
WHEP domain, whereas its mitochondrial isoform possessed an extra N-terminal sequence
consisting of an mitochondrial targeting signal and an appended domain.