Structure studies of the interdomain linker of initiation factor IF-3 and solution structure determination of the N-terminal domain of the ribosomal protein L9.

摘要

Initiation factor IF3 plays an important role in the initiation of protein biosynthesis. It consists of two globular domains linked by a 20 residue solvent exposed linker. The structure studies performed on B. stearothermophilus protein indicate that the linker adopts a helical structure. In contrast, NMR experiments on the E. coli protein indicate that the linker is flexible. Mutation studies on the conserved lysine residues in the linker suggest that the linker region not only is a connecting element but also is important for the ability of IF3 to bind to ribosome. A series of short peptides corresponding to the linker region of IF3 from B. stearothermophilus and E. coli as well as the linker region of chloroplast IF3 from E. gracilis were synthesized to study their secondary structure and stability. Circular dichroism and NMR spectra were used to study the helical content as a function of pH, temperature, peptide concentration and ionic strength. The helical content of the linker fragment from B. stearothermophilus is as high as 50% while the helical content of the E. coli linker is less than 10% at 1°C. The helical content of the linker fragment of chloroplast IF3 from E. gracilis is less than 25%. These results suggest that the different structures observed for the two intact proteins are due to different stabilities of the linker. However, the structure of the linker peptide from different species are very similar when compared at their respective physiological temperature.;The solution structure of the 56 residue N-terminal domain of the ribosomal protein L9 from B. stearothermophilus has been determined by two dimensional 1H-NMR. A total of 366 NOE constraints and 61 dihedral angle restraints as well as 16 H-bond constraints were used to define the structure. The protein folds into a mixed a - b sheet structure with two helices sandwiching a small three stranded b -sheet. The structure is, in general, well defined. The loop between strand one and two appears to adopt a reasonably structured conformation, although it is less well defined than the core of the molecule. The structure of the domain is compared to the structure of the intact protein. Noticeable differences occur in the loop region, in the side chain orientation of the residue involved in RNA binding.;Structure studies of a peptide corresponding to the first 39 residues of N-terminal domain of L9 indicates that a native structure, a so called "ABCD" common motif, can be formed in the absence of the central helix of L9. Though the central helix is not required for the proper folding of the N-terminal domain, it affects the stability of the domain. The structure of the 39 residue peptide is less stable than the structure of the 56 residue peptide.