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

The results reported in the previous paper indicate that the translati onal 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, th e effects of secondary structure in the 5'-untranslated leader of the rbcL mRNA have been examined. Only weak secondary structure can be det ected in the 5'-untranslated leader of the rbcL message by enzymatic a nd computer analysis. Further reduction of the weak secondary structur e of this message by site-directed mutagenesis does not significantly affect the ability of this message to participate in initiation comple x 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 struct ure are much less active in initiation complex formation if the struct ural element introduced is within similar to 10 nucleotides of the sta rt codon. These results suggest that the translational start site in t his chloroplast mRNA is specified by the presence of an AUG codon in a n unstructured or weakly structured region of the mRNA. No specific se quences around the start codon, either upstream or immediately downstr eam, were found to have important information directing the chloroplas t ribosome to the start site of this mRNA.