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.