Role of Arc1p in the modulation of yeast glutamyl-tRNA synthetase activity.
Yeast methionyl-tRNA synthetase (MetRS) and glutamyl-tRNA synthetase (GluRS) possess N-terminal extensions that bind the cofactor Arc1p in trans. The strength of GluRS-Arc1p interaction is high enough to allow copurification of the two macromolecules in a 1:1 ratio, in contrast to MetRS. Deletion analysis from the C-terminal end of the GluRS appendix combined with previous N-terminal deletions of GluRS allows restriction of the Arc1p binding site to the 110-170 amino acid region of GluRS. This region has been shown to correspond to a novel protein-protein interaction domain present in both GluRS and Arc1p but not in MetRS [Galani, K., Grosshans, H., Deinert, K., Hurt, E. C., and Simos, G. (2001) EMBO J. 20, 6889-6898]. The GluRS apoenzyme fails to show significant kinetics of tRNA aminoacylation and charges unfractionated yeast tRNA at a level 10-fold reduced compared to Arc1p- bound GluRS. The K(m) values for tRNA(Glu) measured in the ATP-PP(i) exchange were similar for the two forms of GluRS, whereas k(cat) is increased 2-fold in the presence of Arc1p. Band-shift analysis revealed a 100-fold increase in tRNA binding affinity when Arc1p is bound to GluRS. This increase requires the RNA binding properties of the full-length Arc1p since Arc1p N domain leaves the K(d) of GluRS for tRNA unchanged. Transcripts of yeast tRNA(Glu) were poor substrates for measuring tRNA aminoacylation and could not be used to clarify whether Arc1p has a specific effect on the tRNA charging reaction.[1]References
- Role of Arc1p in the modulation of yeast glutamyl-tRNA synthetase activity. Graindorge, J.S., Senger, B., Tritch, D., Simos, G., Fasiolo, F. Biochemistry (2005) [Pubmed]
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