Initiation factor IF1, the smallest of the three factors, (Cummings and Hershey, 1994), indicating that one or more of the activities of IF1 must The structure of the translational initiation factor be essential for the cell. Maurizio IF1 from Escherichia coli has been determined withSeveral functions have been reported for IF1. These multidimensional NMR spectroscopy. Using 1041 disinclude: (i) the enhancement of the rate of 70S ribosome tance and 78 dihedral constraints, 40 distance geometry dissociation and subunit association (Godefroy-Colburn structures were calculated, which were refined by et al., 1975); (ii) the stimulation of the activity of IF2 restrained molecular dynamics. From this set, 19 strucand IF3 in the formation of the 30S initiation complex tures were selected, having low constraint energy and (Wintermeyer and Gualerzi, 1983; Pon and Gualerzi, few constraint violations. The ensemble of 19 structures 1984); and (iii) the modulation of the interaction of IF2 displays a root-mean-square deviation versus the averwith the ribosome, increasing its affinity for the 30S age of 0.49 Å for the backbone atoms and 1.12 Å for ribosomal subunit when IF1 is bound and indirectly all atoms for residues 6-36 and 46-67. The structure favouring its release when IF1 is ejected (Stringer et al., of IF1 is characterized by a five-stranded β-barrel.1977; Celano et al., 1988). In addition, by binding to the The loop connecting strands three and four contains a A-site of the 30S ribosomal subunit, IF1 may contribute short 3 10 helix but this region shows considerably to the fidelity of the selection of the initiation site of the higher flexibility than the β-barrel. The fold of IF1 is mRNA (Moazed et al., 1995). very similar to that found in the bacterial cold shock Equilibrium binding studies have shown that IF1 binds proteins CspA and CspB, the N-terminal domain of to the 30S ribosomal subunit in a 1:1 ratio (Zucker and aspartyl-tRNA synthetase and the staphylococcal Hershey, 1986;Celano et al., 1988). The binding affinity nuclease, and can be identified as the oligomer-binding depends strongly upon the ionic strength and upon the motif. Several proteins of this family are nucleic acidpresence of IF2 and IF3 which increase its affinity for the binding proteins. This suggests that IF1 plays its role ribosome; the K a ranging from 5ϫ10 5 to 2.5ϫ10 8 M -1 . in the initiation of protein synthesis by nucleic acid Interaction with 50S ribosomal subunits was also observed, interactions. Specific changes of NMR signals of IF1 but the affinity is considerably lower than for the 30S upon titration with 30S ribosomal subunit identifies ribosomal subunits. Stable interaction with 70S ribosomes several residues that are involved in the interaction has never been observed; in fact, the addition of 50S to with ribosomes.
Titrations of Escherichia coli translation initiation factor IF3, isotopically labeled with 15 N, with 30S ribosomal subunits were followed by NMR by recording two-dimensional ( 15 N, 1 H)-HSQC spectra. In the titrations, intensity changes are observed for cross peaks belonging to amides of individual amino acids. At low concentrations of ribosomal subunits, only resonances belonging to amino acids of the C-domain of IF3 are affected, whereas all those attributed to the N-domain are still visible. Upon addition of a larger amount of 30S subunits cross peaks belonging to residues of the N-terminal domain of the protein are also selectively affected.Our results demonstrate that the two domains of IF3 are functionally independent, each interacting with a different affinity with the ribosomal subunits, thus allowing the identification of the individual residues of the two domains involved in this interaction. Overall, the C-domain interacts with the 30S subunits primarily through some of its loops and a-helices and the residues involved in ribosome binding are distributed rather symmetrically over a fairly large surface of the domain, while the N-domain interacts mainly via a small number of residues distributed asymmetrically in this domain.The spatial organization of the active sites of IF3, emerging through the comparison of the present data with the previous chemical modification and mutagenesis data, is discussed in light of the ribosomal localization of IF3 and of the mechanism of action of this factor.
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