Initiator tRNA binding by e/aIF5B, the eukaryotic/archaeal homologue of bacterial initiation factor IF2.
To carry initiator Met-tRNA(i)(Met) to the small ribosomal subunit, eukaryal and archaeal cells use a heterotrimeric factor called e/aIF2. These cells also possess a homologue of bacterial IF2 called e/aIF5B. Several results indicate that the mode of action of e/aIF5B resembles some function of bacterial IF2. The e/aIF5B factor promotes the joining of ribosomal subunits. Moreover, there is genetic evidence that the factor participates in the binding of initiator tRNA to the small ribosomal subunit. However, up to now, an interaction between e/aIF5B and initiator tRNA was not evidenced. In this study, we use an assay based on protection of aminoacyl-tRNA against spontaneous deacylation to demonstrate that archaeal aIF5B indeed can interact with initiator tRNA. In complex formation, aIF5B shows specificity toward the methionyl moiety of the ligand. The complex between Saccharomyces cerevisiae eIF5B and methionylated initiator tRNA is less stable than that formed with aIF5B. In addition, this complex is almost indifferent to the side chain of the esterified amino acid. These results support the idea that, beyond the channeling of Met-tRNA(i)(Met) to the 40S subunit by e/aIF2, e/aIF5B comes to interact with initiator tRNA on the ribosome. Recognition of an aminoacylated tRNA species at this site would then allow translation to begin. In the case of archaea, this checkpoint would also include the verification of the presence of a methionine at the P site.
Guillon
Laurent
L
Laboratoire de Biochimie, Unité Mixte de Recherche 7654, CNRS-Ecole Polytechnique, F-91128 Palaiseau Cedex, France.
Schmitt
Emmanuelle
E
Blanquet
Sylvain
S
Mechulam
Yves
Y
D019843:Archaeal Proteins / Q000378:metabolism; D001665:Binding Sites; D010448:Peptide Initiation Factors / Q000378:metabolism* / Q000502:physiology; D039881:Prokaryotic Initiation Factor-2; D012358:RNA, Transfer, Met / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism / Q000502:physiology; D040681:Structural Homology, Protein
D019843:Archaeal Proteins; C118735:IF2 protein, archaeal; D010448:Peptide Initiation Factors; D039881:Prokaryotic Initiation Factor-2; D012358:RNA, Transfer, Met; D029701:Saccharomyces cerevisiae Proteins