EFFECT OF THE SECONDARY STRUCTURE IN THE EUGLENA-GRACILIS CHLOROPLAST RIBULOSE-BISPHOSPHATE CARBOXYLASE/OXYGENASE MESSENGER-RNA ON TRANSLATIONAL INITIATION

The results reported in the previous paper indicate that the translational start site of the Euglena gracilis chloroplast mRNA for the large subunit of ribulose-bisphosphate carboxylase/oxygenase (rbcL) is not defined by primary sequence elements (Koo, J. S., and Spremulli, L. L. (1994) J. Biol. Chem. 269, 7494-7500). In the work presented here, the effects of secondary structure in the 5'-untranslated leader of the rbcL mRNA have been examined. Only weak secondary structure can be detected in the 5'-untranslated leader of the rbcL message by enzymatic and computer analysis. Further reduction of the weak secondary structure of this message by site-directed mutagenesis does not significantly affect the ability of this message to participate in initiation complex formation. The secondary structure near the translational start site was increased by the introduction of an inverted repeat sequence and by site-directed mutagenesis. Messages with increased secondary structure are much less active in initiation complex formation if the structural element introduced is within similar to 10 nucleotides of the start codon. These results suggest that the translational start site in this chloroplast mRNA is specified by the presence of an AUG codon in an unstructured or weakly structured region of the mRNA. No specific sequences around the start codon, either upstream or immediately downstream, were found to have important information directing the chloroplast ribosome to the start site of this mRNA.