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 meth ionyl-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 o f this bipartite RNA-binding domain are functionally distinct. Analysis of MetRS mutant enzymes revealed that the HTH motif is more specifically endow ed with a tRNA-sequestering activity and confers on MetRS a rate-limiting d issociation of aminoacylated tRNA. Elongation factor EF-1 alpha enhanced th e turnover in the aminoacylation reaction. In contrast, the LRC region is m ost probably involved in accelerating the association step of deacylated tR NA. These two nonredundant RNA-binding motifs strengthen tRNA binding by th e synthetase. The native form of MetRS, containing the C-terminal RNA-bindi ng 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-1 alpha -assisted dissociation of Met-tRNA(met). The refore, the eukaryotic-specific C-domain of human MetRS may have a dual fun ction. It may ensure an efficient capture of tRNA(met) under conditions of suboptimal deacylated tRNA concentration prevailing in vivo, and may instig ate direct transfer of aminoacylated tRNA from the synthetase to elongation factor EF-1 alpha.