ISOLATION AND CHARACTERIZATION OF CDNA CLONES FOR CHLOROPLAST TRANSLATIONAL INITIATION FACTOR-III FROM EUGLENA-GRACILIS

A complete cDNA clone encoding Euglena gracilis chloroplast translatio nal initiation factor 3 (IF-3chl) has been obtained. Analysis of the s equence indicates that the IF-3chl mRNA contains the spliced leader fo und at the 5' end of nuclear encoded mRNAs in E. gracilis. The open re ading frame for IF-3chl encodes a 537-amino acid protein. IF-3chl appe ars to be divided into four domains. The first 140 amino acids corresp ond to a transit peptide required for the import of IF-3chl into the c hloroplast. 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 n o homology to other known proteins. It begins with a stretch of 35 res idues, of which about 30% are proline. Downstream from this region is a stretch of about 25 amino acids with a repeating (GX)n motif followe d by a very acidic region. The third domain comprises a region of abou t 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 o f 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 presen t in approximately equal amounts in both dark- and light-grown cells, suggesting that the light-dependent induction of IF-3chl activity is p ost-transcriptional.