Functional molecular mapping of archaeal translation initiation factor 2.
Eukaryotic and archaeal initiation factors 2 (e/aIF2) are heterotrimeric proteins (alphabetagamma) carrying methionylated initiator tRNA to the small subunit of the ribosome. The three-dimensional structure of aIF2gamma from the Archaea Pyrococcus abyssi was previously solved. This subunit forms the core of the heterotrimer. The alpha and beta subunits bind the gamma, but do not interact together. aIF2gamma shows a high resemblance with elongation factor EF1-A. In this study, we characterize the role of each subunit in the binding of the methionylated initiator tRNA. Studying various aminoacyl-tRNA ligands shows that the methionyl group is a major determinant for recognition by aIF2. aIF2gamma alone is able to specifically bind Met-tRNAiMet, although with a reduced affinity as compared with the intact trimer. Site-directed mutagenesis confirms a binding mode of the tRNA molecule similar to that observed with the elongation factor. Under our assay conditions, aIF2beta is not involved in the docking of the tRNA molecule. In contrast, aIF2alpha provides the heterotrimer its full tRNA binding affinity. Furthermore, the isolated C-domain of aIF2alpha is responsible for binding of the alpha subunit to gamma. This binding involves an idiosyncratic loop of domain 2 of aIF2gamma. Association of the C-domain of aIF2alpha to aIF2gamma is enough to retrieve the binding affinity of tRNA for aIF2. The N-terminal and central domains of aIF2alpha do not interfere with tRNA binding. However, the N-domain of aIF2alpha interacts with RNA unspecifically. Based on this property, a possible contribution of aIF2alpha to formation of a productive complex between aIF2 and the small ribosomal subunit is envisaged.
Yatime
Laure
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 / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D010449:Peptide Mapping; D039881:Prokaryotic Initiation Factor-2 / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D043903:Pyrococcus abyssi / Q000737:chemistry*; D012343:RNA, Transfer / Q000032:analysis; D013329:Structure-Activity Relationship
D019843:Archaeal Proteins; D039881:Prokaryotic Initiation Factor-2; D012343:RNA, Transfer