Identification of the ribosome binding sites of translation initiation factor IF3 by multidimensional heteronuclear NMR spectroscopy

Abstract

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.

The Search and Its Outcome: High-Resolution Structures of Ribosomal Particles from Mesophilic, Thermophilic, and Halophilic Bacteria at Various Functional States

“…Using crystals of T30S in complex with IF3C, IF3C binds to the 30S particle at the upper end of the platform on the solvent side (Figure 6), close to the anti-SD region of the 16S rRNA (50). This location reconfirms the results of NMR and mutagenesis of the IF3 molecule (55) and is compatible with the effect of the double mutations 1503, 1531 (19). It is also consistent with almost all the crosslinks, footprints, and protection patterns reported for the E. coli system (38,52).…”

“…Its long terminus tails are more ordered in the tungstenated or IF3C-bound T30S than in the Co-hexamine-treated small subunits. These tails appear to act as tentacles that enhance the binding of IF3C, consistent with the firm binding of this domain to the ribosome (55,67). They are also capable of binding the IF3C mimic, namely the W18 cluster (Figure 7).…”

The Molecular Basis of Distinct Aggregation Pathways of Islet Amyloid Polypeptide

“…At room temperature and pH 6.5 all of our samples showed a time-dependent decrease of cross-peak intensities likely due to the slow sequestering of soluble IAPP into insoluble aggregates. However, due to pseudo-equilibrium between the association and dissociation of IAPP to and from aggregates, excessive transverse relaxation of spins at the binding interface might ensue (33)(34). Thus, using the concomitant relative decrease in intensities of the corresponding cross-peaks, preferential binding of individual residues to growing surfaces of aggregates may be established (33)(34).…”

Preliminary characterization by X-ray diffraction and Raman spectroscopy of a crystalline complex ofBacillus stearothermophilusinitiation factor 2 C-domain and fMet-tRNAfMet

“…Until now, neither the threedimensional structure of IF2 nor of its complex with fMet-tRNA fMet were available. Threedimensional structures are known for the two other prokaryotic translation initiation factors, IF1 (Sette et al, 1997) and IF3 (Biou et al, 1995).…”