title: Translation initiation factor IF2 interacts with the 30 S ribosomal subunit via two separate binding sites.
abstract: The functional properties of the two natural forms of Escherichia coli translation initiation factor IF2 (IF2alpha and IF2beta) and of an N-terminal deletion mutant of the factor (IF2DeltaN) lacking the first 294 residues, corresponding to the entire N-terminal domain, were analysed comparatively. The results revealed that IF2alpha and IF2beta display almost indistinguishable properties, whereas IF2DeltaN, although fully active in all steps of the translation initiation pathway, displays functional activities having properties and requirements distinctly different from those of the intact molecule. Indeed, binding of IF2DeltaN to the 30 S subunit, IF2DeltaN-dependent stimulation of fMet-tRNA binding to the ribosome and of initiation dipeptide formation strongly depend upon the presence of IF1 and GTP, unlike with IF2alpha and IF2beta. The present results indicate that, using two separate active sites, IF2 establishes two interactions with the 30 S ribosomal subunit which have different properties and functions. The first site, located in the N domain of IF2, is responsible for a high-affinity interaction which "anchors" the factor to the subunit while the second site, mainly located in the beta-barrel module homologous to domain II of EF-G and EF-Tu, is responsible for the functional ("core") interaction of IF2 leading to the decoding of fMet-tRNA in the 30 S subunit P-site. The first interaction is functionally dispensable, sensitive to ionic-strength variations and essentially insensitive to the nature of the guanosine nucleotide ligand and to the presence of IF1, unlike the second interaction which strongly depends upon the presence of IF1 and GTP.
authors.affiliation: Laboratory of Genetics, Department of Biology, University of Camerino, 62032 Camerino (MC), Italy.
authors.forename: Enrico
authors.initials: E
authors.lastname: Caserta
authors.forename: Jerneja
authors.initials: J
authors.lastname: Tomsic
authors.forename: Roberto
authors.initials: R
authors.lastname: Spurio
authors.forename: Anna
authors.initials: A
authors.lastname: La Teana
authors.forename: Cynthia L
authors.initials: CL
authors.lastname: Pon
authors.forename: Claudio O
authors.initials: CO
authors.lastname: Gualerzi
mesh_terms: D000926:Anticodon / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003062:Codon / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli; D006150:Guanine Nucleotides / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D050505:Mutant Proteins / Q000378:metabolism; D039881:Prokaryotic Initiation Factor-2 / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D012358:RNA, Transfer, Met / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D000926:Anticodon; D003062:Codon; D006150:Guanine Nucleotides; D008024:Ligands; D050505:Mutant Proteins; D039881:Prokaryotic Initiation Factor-2; D012358:RNA, Transfer, Met