Regulation of the activity of chloroplast translational initiation factor 3 by NH2- and COOH-terminal extensions

The mature form of the chloroplast translational initiation factor 3 (IF3(chl)) from Euglena gracilis consists of an internal region homologous to prokaryotic IF3 flanked by long NH2- and COOH-terminal extensions. Sequences in these extensions reduce the activity of the homology domain in promoting initiation complex formation with chloroplast mRNAs and 30 S ribosomal sub units. A series of deletions of the NH2- and COOH-terminal extensions of IF3(chl) were constructed and tested for their effects on the activity of the homology domain. About half of the inhibitory effect arises from sequences within 9 residues of the junction between the NH2-terminal extension and the homology domain. The remaining inhibitory effect is the result of sequences in the COOH-terminal extension. The equilibrium constant governing the binding of the homology domain of IF3(chl) to 30 S subunits is estimated to be 1.3 x 10(7) M-1. Sequences close to the junction of the NH2-terminal extension and the homology domain reduce this binding constant about 10-fold. Sequences in the COOH-terminal extension have a similar negative effect. The negative effects of these two regions are cumulative, resulting in a 100-fold reduction of the binding constant. The 9 residues at the NH2-terminal extension effectively prevent the proofreading activity of IF3(chl). The entire COOH-terminal extension reduces the proofreading ability by about half. These results are discussed in terms of the proposed three-dimensional structure of the homology domain of IF3(chl).