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KMID : 0603820070130020105
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Journal of Experimental & Biomedical Science
2007 Volume.13 No. 2 p.105 ~ p.110
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Aspartyl-tRNA Synthetase from Acidithiobacillus ferrooxidans Aspartylates Both tRNA {Asp} and tRNA {Asn}
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Keem Joo-Oak
Choi Soon-Yong
Koh Suk-Hoon
Hyun Sung-Hee
Min Bok-Kee
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Abstract
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Aspartyl-tRNA synthetase (AspRS) exists in two different forms with respect to tRNA recognition. The discriminating enzyme (D-AspRS) recognizes only tRNA {Asp}, while the non-discriminating one (ND-AspRS) also recognizes tRNA {Asn} and therefore forms both Asp-tRNA {Asn} and Asp-tRNA {Asp}. Plus primary sequence distinguishes two general groups of AspRS. There is a predominantly bacterial-type, larger AspRS (about 580 aa) in addition to a shorter archaeal/eukaryotic type (about 430 aa). In vivo data made clear that discriminating and non-discriminating enzymes exist in both groups. The determinants in the protein sequence responsible for tRNA discrimination are not hewn. The AspRS from Acidithiobacillus ferrooxidans might be suggested ND-AspRS fur missing of AsnRS in genomic sequencing data. Therefore, we analyzed the AspRS from A. ferrooxidans with in vitro aminoacylation assay with E. coli unfractionated tRNA, in vivo missense suppression assay with tipA34 mutant and Northern hybridization with probes which were specific with tRNA {Asp} or tRNA {Asn}. The AspRS from A. ferrooxidans produced more Asp-tRNA than that from E. coli. Only aspS gene from A. ferrooxidans suppressed trpA34 strain in minimal media without tryptophan. Only AspRS from A. ferrooxidans showed mischarged Asp-tRNA {Asn} band. Therefore, AspRS from A. ferrooxidans is definitely ND-AspRS.
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KEYWORD
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Nondiscriminating aspartyl-tRNA synthetase, Discriminating aspartyl-tRNA synthetase, Mischarging, Aminoacylation, missense suppression
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