ON THE GLOBAL ARCHITECTURE OF INITIATION-FACTOR IF3 - A COMPARATIVE-STUDY OF THE LINKER REGIONS FROM THE ESCHERICHIA-COLI PROTEIN AND THE BACILLUS-STEAROTHERMOPHILUS PROTEIN

Initiation factor IF3 is a protein involved in the initiation stage of protein synthesis. It consists of two global domains linked by a 20 r esidue long, solvent-exposed Linker. Recently, the structure of the N and C-terminal domains of the Bacillus stearothermophilus protein have been solved by X-ray crystallography and the structure of the intact Escherichia coli protein has been studied by NMR. These two studies ha ve led to apparently contradictory models for the domain organization of IF3. The NMR study of the E. coli protein indicates that the linker region is flexible, while the studies of the isolated N and C-termina l domains of the B. stearothermophilus protein suggest that the linker forms a rigid helical rod. Ln order to resolve this discrepancy, a se t of peptides corresponding to the linker regions of the B. stearother mophilus and the E. coli protein were synthesized. Circular dichroism and NMR spectroscopy were used to study the helical content as a uncti on of pH, temperature, peptide concentration and ionic strength. Both peptides are monomeric. The estimated helical content of the linker fr agment from B. stearothermophilus is 68% at high pH and 1 degrees C. T he measured helicity decreases to 53% at pH 7.0 and 1 degrees C. In co ntrast, the peptide corresponding to the E. coli IF3 linker region is largely unstructured with a maximum helical content of 15% at high ph and only 8% at pH 7.0, 1 degrees C. These results suggest that the dif ferent structures observed for the two intact proteins may be due to t he different intrinsic stability of the two linker peptides. The helic al content of the two linker peptides is however, much closer when the peptides are compared at the respective temperatures of optimum growt h for E. coli and B. stearothermophilus (3% versus 17%). The pH and io nic strength dependence of the helical content of the B. stearothermop hilus peptide demonstrates that side-chain/side-chain interactions pla y an important role in stabilizing the helical structure. In addition, studies with mutant peptides show that the first Asp residue in the l inker sequence help to stabilize the helix via an N-capping interactio n. (C) 1998 Academic Press Limited.