GTP-dependent recognition of the methionine moiety on initiator tRNA by translation factor eIF2.
Eukaryotic translation initiation factor 2 (eIF2) is a G-protein that functions as a central switch in the initiation of protein synthesis. In its GTP-bound state it delivers the methionyl initiator tRNA (Met-tRNA(i)) to the small ribosomal subunit and releases it upon GTP hydrolysis following the recognition of the initiation codon. We have developed a complete thermodynamic framework for the assembly of the Saccharomyces cerevisiae eIF2.GTP.Met-tRNA(i) ternary complex and have determined the effect of the conversion of GTP to GDP on eIF2's affinity for Met-tRNA(i) in solution. In its GTP-bound state the factor forms a positive interaction with the methionine moiety on Met-tRNA(i) that is disrupted when GTP is replaced with GDP, while contacts between the factor and the body of the tRNA remain intact. This positive interaction with the methionine residue on the tRNA may serve to ensure that only charged initiator tRNA enters the initiation pathway. The toggling on and off of the factor's interaction with the methionine residue is likely to play an important role in the mechanism of initiator tRNA release upon initiation codon recognition. In addition, we show that the conserved base-pair A1:U72, which is known to be a critical identity element distinguishing initiator from elongator methionyl tRNA, is required for recognition of the methionine moiety by eIF2. Our data suggest that a role of this base-pair is to orient the methionine moiety on the initiator tRNA in its recognition pocket on eIF2.
Kapp
Lee D
LD
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe Street 625 WBSB, Baltimore, MD 21205-2185, USA.
Lorsch
Jon R
JR
D020029:Base Pairing; D001483:Base Sequence; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008715:Methionine / Q000378:metabolism*; D008969:Molecular Sequence Data; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012358:RNA, Transfer, Met / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D013816:Thermodynamics
D015852:Eukaryotic Initiation Factor-2; D012331:RNA, Fungal; D012358:RNA, Transfer, Met; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D008715:Methionine