ANALYSIS OF THE TRANSLATIONAL INITIATION REGION ON THE EUGLENA-GRACILIS CHLOROPLAST RIBULOSE-BISPHOSPHATE CARBOXYLASE OXYGENASE (RBCL) MESSENGER-RNA/

The chloroplast mRNAs from Euglena gracilis fall into two classes. One group of mRNAs from this organelle contains a Shine-Dalgarno sequence 5' to the start codon, while the other group of mRNAs does not have a conserved sequence signal in the 5'-untranslated region. To investiga te the start signals for E. gracilis chloroplast mRNAs that do not car ry a Shine-Dalgarno sequence, 30 S initiation complex formation has be en studied using a series of transcripts carrying the wild-type transl ational start site of ribulose-bisphosphate carboxylase/oxygenase (rbc L) or mutated derivatives of this site. Mutation of the start codon of the rbcL gene indicates that the chloroplast 30 S subunit is recogniz ing only the correct AUG codon. The analysis of the messages from a se ries of deletion mutants shows that a minimum of Lambda 20 residues 5' to the AUG codon is required for activity. Maximal activity requires the full 55-base leader sequence. Surprisingly, a transcript carrying the inverse complement of 48 bases in the leader is Lambda 60% as acti ve as the wild-type message in promoting initiation complex formation. Introduction of a Shine-Dalgarno sequence in the 5'-leader increases the activity of the mRNA only Lambda 1.4-2-fold. The presence of an ol igodeoxynucleotide containing a strong Shine-Dalgarno sequence does no t significantly inhibit the formation of initiation complexes at the r bcL start site. Similar results are obtained when initiation complexes are formed with initiation factors from either E. gracilis chloroplas ts or Escherichia coli.