The appended C-domain of human methionyl-tRNA synthetase has a tRNA-sequestering function.
An ancillary RNA-binding domain is appended to the C-terminus of human methionyl-tRNA synthetase. It comprises a helix-turn-helix (HTH) motif related to the repeated units of the linker region of bifunctional glutamyl-prolyl-tRNA synthetase, and a specific C-terminal KGKKKK lysine-rich cluster (LRC). Here we show by gel retardation and tRNA aminoacylation experiments that these two regions are important for tRNA binding. However, the two pieces of this bipartite RNA-binding domain are functionally distinct. Analysis of MetRS mutant enzymes revealed that the HTH motif is more specifically endowed with a tRNA-sequestering activity and confers on MetRS a rate-limiting dissociation of aminoacylated tRNA. Elongation factor EF-1alpha enhanced the turnover in the aminoacylation reaction. In contrast, the LRC region is most probably involved in accelerating the association step of deacylated tRNA. These two nonredundant RNA-binding motifs strengthen tRNA binding by the synthetase. The native form of MetRS, containing the C-terminal RNA-binding domain, behaves as a processive enzyme; release of the reaction product is not spontaneous, but may be synchronized with the subsequent step of the tRNA cycle through EF-1alpha-assisted dissociation of Met-tRNA(Met). Therefore, the eukaryotic-specific C-domain of human MetRS may have a dual function. It may ensure an efficient capture of tRNA(Met) under conditions of suboptimal deacylated tRNA concentration prevailing in vivo, and may instigate direct transfer of aminoacylated tRNA from the synthetase to elongation factor EF-1alpha.
Kaminska
M
M
Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, 1 Avenue de la Terrasse, 91190 Gif-sur-Yvette, France.
Shalak
V
V
Mirande
M
M
D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases; D001665:Binding Sites; D019077:Helix-Turn-Helix Motifs; D006801:Humans; D008718:Methionine-tRNA Ligase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D020648:Peptide Elongation Factor 1 / Q000378:metabolism; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D012343:RNA, Transfer / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012358:RNA, Transfer, Met / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D017386:Sequence Homology, Amino Acid
D020648:Peptide Elongation Factor 1; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; D016601:RNA-Binding Proteins; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; C415462:glutamyl-prolyl-tRNA synthetase; D008718:Methionine-tRNA Ligase