Heteronuclear NMR studies of E-coli translation initiation factor IF3. Evidence that the inter-domain region is disordered in solution

Initiation factor IF3 from Escherichia coli plays a critical role in the selection of the correct initiation codon. This protein is composed of two domains, connected by a lysine-rich hydrophilic linker. The conformation of native IF3 was investigated by heteronuclear NMR spectroscopy. The two domains are independent and show little or no interaction. Heteronuclear relaxation studies of a sample selectively labelled on lysine residues demonstrates that the inter-domain Linker is highly flexible, exhibiting increased N-15 T-2 values and negative H-1{N-15} nuclear Overhause effects over a length of at least eight residues. Analysis of the rotational correlation times further shows that the motions of the two domains are most Likely uncorrelated. The inter-domain linker thus displays almost totally unrestricted motions. Accordingly, the amide protons in the central region are shown to be in fast exchange with water. Such a high degree of flexibility of the inter-domain linker might be required for IF3 domains to interact with distant regions of the ribosome. (C) 1997 Academic Press Limited.