Isolation and characterization of cDNA clones for chloroplast translational initiation factor-3 from Euglena gracilis

A complete cDNA clone encoding Euglena gracilis chloroplast translational initiation factor 3 (IF-3chl) has been obtained. Analysis of the sequence indicates that the IF-3chl mRNA contains the spliced leader found at the 5' end of nuclear encoded mRNAs in E. gracilis. The open reading frame for IF-3chl encodes a 537-amino acid protein. IF-3chl appears to be divided into four domains. The first 140 amino acids correspond to a transit peptide required for the import of IF-3chl into the chloroplast. The mature form of IF-3chl encompasses domains 2-4 and is about twice the size of Escherichia coli IF-3. The second domain has no homology to other known proteins. It begins with a stretch of 35 residues, of which about 30% are proline. Downstream from this region is a stretch of about 25 amino acids with a repeating (GX)n motif followed by a very acidic region. The third domain comprises a region of about 175 residues and has between 31 and 37% homology to the IF-3s found in other organisms. The IF-3 homology domain is followed by an acidic region which has no detectable homology to other sequences. Analysis of E. gracilis genomic DNA suggests that there are about four copies of the IF-3chl gene, one of which is probably a pseudogene. The activity of IF-3chl is inducible by light. However, the IF-3chl mRNA is present in approximately equal amounts in both dark- and light-grown cells, suggesting that the light-dependent induction of IF-3chl activity is post-transcriptional.