Association between Archaeal prolyl- and leucyl-tRNA synthetases enhances tRNA(Pro) aminoacylation.
Aminoacyl-tRNA synthetase-containing complexes have been identified in different eukaryotes, and their existence has also been suggested in some Archaea. To investigate interactions involving aminoacyl-tRNA synthetases in Archaea, we undertook a yeast two-hybrid screen for interactions between Methanothermobacter thermautotrophicus proteins using prolyl-tRNA synthetase (ProRS) as the bait. Interacting proteins identified included components of methanogenesis, protein-modifying factors, and leucyl-tRNA synthetase (LeuRS). The association of ProRS with LeuRS was confirmed in vitro by native gel electrophoresis and size exclusion chromatography. Determination of the steady-state kinetics of tRNA(Pro) charging showed that the catalytic efficiency (k(cat)/K(m)) of ProRS increased 5-fold in the complex with LeuRS compared with the free enzyme, whereas the K(m) for proline was unchanged. No significant changes in the steady-state kinetics of LeuRS aminoacylation were observed upon the addition of ProRS. These findings indicate that ProRS and LeuRS associate in M. thermautotrophicus and suggest that this interaction contributes to translational fidelity by enhancing tRNA aminoacylation by ProRS.
Praetorius-Ibba
Mette
M
Department of Microbiology, The Ohio State University, Columbus, Ohio 43210-1292, USA. ibba.1@osu.edu
Rogers
Theresa E
TE
Samson
Rachel
R
Kelman
Zvi
Z
Ibba
Michael
M
D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry / Q000502:physiology*; D002845:Chromatography; D002850:Chromatography, Gel; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D007935:Leucine-tRNA Ligase / Q000737:chemistry / Q000502:physiology*; D017013:Methanobacteriaceae / Q000378:metabolism; D010641:Phenotype; D011485:Protein Binding; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D012361:RNA, Transfer, Pro / Q000737:chemistry*; D013696:Temperature; D020798:Two-Hybrid System Techniques
D018076:DNA, Complementary; D012333:RNA, Messenger; D012361:RNA, Transfer, Pro; D006639:Histidine; D000604:Amino Acyl-tRNA Synthetases; C100134:prolyl T RNA synthetase; D007935:Leucine-tRNA Ligase