Titrations of Escherichia coli translation initiation
factor IF3, isotopically labeled with 15N,
with 30S ribosomal subunits were followed by NMR by recording
two-dimensional (15N,1H)-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 α-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.