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.
Graindorge
Jean-Sébastien
JS
UPR no. 9002 du CNRS, Institut de Biologie Moleculaire et Cellulaire du CNRS, 15 rue Rene Descartes, 67084 Strasbourg Cedex, France.
Senger
Bruno
B
Tritch
Denis
D
Simos
George
G
Fasiolo
Franco
F
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D049148:Aminoacylation; D001483:Base Sequence; D011756:Diphosphates / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation; D015966:Gene Expression Regulation, Fungal; D005975:Glutamate-tRNA Ligase / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012352:RNA, Transfer, Glu / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic
C102145:ARC1 protein, S cerevisiae; D011756:Diphosphates; D010446:Peptide Fragments; D012331:RNA, Fungal; D012352:RNA, Transfer, Glu; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D005975:Glutamate-tRNA Ligase