Cys-tRNA(Pro) editing by Haemophilus influenzae YbaK via a novel synthetase.YbaK.tRNA ternary complex.
Aminoacyl-tRNA synthetases are multidomain enzymes that often possess two activities to ensure translational accuracy. A synthetic active site catalyzes tRNA aminoacylation, while an editing active site hydrolyzes mischarged tRNAs. Prolyl-tRNA synthetases (ProRS) have been shown to misacylate Cys onto tRNA(Pro), but lack a Cys-specific editing function. The synthetase-like Haemophilus influenzae YbaK protein was recently shown to hydrolyze misacylated Cys-tRNA(Pro) in trans. However, the mechanism of specific substrate selection by this single domain hydrolase is unknown. Here, we demonstrate that YbaK alone appears to lack specific tRNA recognition capabilities. Moreover, YbaK cannot compete for aminoacyl-tRNAs in the presence of elongation factor Tu, suggesting that YbaK acts before release of the aminoacyl-tRNA from the synthetase. In support of this idea, cross-linking studies reveal the formation of binary (ProRS.YbaK) and ternary (ProRS.YbaK.tRNA) complexes. The binding constants for the interaction between ProRS and YbaK are 550 nM and 45 nM in the absence and presence of tRNA(Pro), respectively. These results suggest that the specificity of trans-editing by YbaK is ensured through formation of a novel ProRS.YbaK.tRNA complex.
An
Songon
S
Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Musier-Forsyth
Karin
K
D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry; D001426:Bacterial Proteins / Q000378:metabolism / Q000502:physiology*; D001665:Binding Sites; D002384:Catalysis; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000378:metabolism; D006193:Haemophilus influenzae / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D010444:Peptide Elongation Factor Tu / Q000737:chemistry; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D017393:RNA Editing; D012343:RNA, Transfer / Q000737:chemistry*; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry*; D013379:Substrate Specificity; D013997:Time Factors; D046249:Transfer RNA Aminoacylation
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D012346:RNA, Transfer, Amino Acyl; C481278:YbaK protein, Haemophilus influenzae; D012343:RNA, Transfer; D010444:Peptide Elongation Factor Tu; D000604:Amino Acyl-tRNA Synthetases; D003545:Cysteine