Eukaryotic translation initiation factor 5 (eIF5) acts as a classical GTPase-activator protein.
GTP hydrolysis occurs at several specific stages during the initiation, elongation, and termination stages of mRNA translation. However, it is unclear how GTP hydrolysis occurs; it has previously been suggested to involve a GTPase active center in the ribosome, although proof for this is lacking. Alternatively, it could involve the translation factors themselves, e.g., be similar to the situation for small G in which the GTPase active site involves arginine residues contributed by a further protein termed a GTPase-activator protein (GAP). During translation initiation in eukaryotes, initiation factor eIF5 is required for hydrolysis of GTP bound to eIF2 (the protein which brings the initiator Met-tRNA(i) to the 40S subunit). Here we show that eIF5 displays the hallmarks of a classical GAP (e.g., RasGAP). Firstly, its interaction with eIF2 is enhanced by AlF(4)(-). Secondly, eIF5 possesses a conserved arginine (Arg15) which, like the "arginine fingers" of classical GAPs, is flanked by hydrophobic residues. Mutation of Arg15 to methionine abolishes the ability of eIF5 either to stimulate GTP hydrolysis or to support mRNA translation in vitro. Mutation studies suggest that a second conserved arginine (Arg48) also contributes to the GTPase active site of the eIF2.eIF5 complex. Our data thus show that eIF5 behaves as a classical GAP and that GTP hydrolysis during translation involves proteins extrinsic to the ribosome. Indeed, inspection of their sequences suggests that other translation factors may also act as GAPs.
Paulin
F E
FE
School of Life Sciences, University of Dundee, DD1 5EH, Dundee, Scotland, United Kingdom.
Campbell
L E
LE
O'Brien
K
K
Loughlin
J
J
Proud
C G
CG
D000595:Amino Acid Sequence; D004789:Enzyme Activation; D040061:Eukaryotic Initiation Factor-5; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D040061:Eukaryotic Initiation Factor-5; D010448:Peptide Initiation Factors; D020558:GTP Phosphohydrolases