ANALYSIS OF THE ROLE OF THE SHINE-DALGARNO SEQUENCE AND MESSENGER-RNASECONDARY STRUCTURE ON THE EFFICIENCY OF TRANSLATIONAL INITIATION IN THE EUGLENA-GRACILIS CHLOROPLAST ATPH MESSENGER-RNA

Chloroplast mRNAs in Euglena gracilis fall into two classes. One class has a Shine-Dalgarno sequence 5' to the AUG start codon while the oth er group of mRNAs does not have any conserved sequence elements near t he start codon. The chloroplast mRNA encoding the atpH gene has been s elected as an example of a message which has a Shine-Dalgarno sequence (GGAGUU) located in the initiation region. Mutations in the Shine-Dal garno sequence result in 2-5-fold reductions in the efficiency of the message in initiation complex formation depending on the precise mutat ion. Secondary structure mapping of the initiation region of the atpH mRNA suggests that a number of mRNA conformations are present in an eq uilibrium mixture. Mutations in the Shine-Dalgarno sequence had little effect on the overall structure of the mRNA Directed mutations which place the Shine-Dalgarno sequence into a region of higher secondary st ructure drastically reduce the activity of the message. The monocistro nic form of the atpH mRNA has a 5'-untranslated leader region slightly over 70 nucleotides in length. Deletions in the leader suggest that a minimum length of 10 bases 5' to the start codon is important for act ivity in initiation. The presence of the full leader increased efficie ncy in initiation about 2-fold compared to messages with leaders 25-40 residues in length.