Analysis of the role of the Shine-Dalgarno sequence and mRNA secondary structure on the efficiency of translational initiation in the Euglena gracilis chloroplast atpH mRNA

L Betts,L L Spremulli

doi:10.1016/s0021-9258(18)47216-x

Abstract

Chloroplast mRNAs in Euglena gracilis fall into two classes. One class has a Shine-Dalgarno sequence 5' to the AUG start codon while the other group of mRNAs does not have any conserved sequence elements near the start codon. The chloroplast mRNA encoding the atpH gene has been selected as an example of a message which has a Shine-Dalgarno sequence (GGAGUU) located in the initiation region. Mutations in the Shine-Dalgarno sequence result in 2-5-fold reductions in the efficiency of the message in initiation complex formation depending on the precise mutation. Secondary structure mapping of the initiation region of the atpH mRNA suggests that a number of mRNA conformations are present in an equilibrium 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 structure drastically reduce the activity of the message. The monocistronic 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 activity in initiation. The presence of the full leader increased efficiency in initiation about 2-fold compared to messages with leaders 25-40 residues in length.

Highlights

A survey of higher plant chloroplast mRNA translational thestartcodon.Thechloroplast mRNA encoding the start sites has shown that over 90% have a sequence compleatpH gene has been selecteads an example oaf message mentary to thcehloroplast 16S rRNA sequence [7,8]
These results strongly suggest that the in vitro system used hereaccurately recognizes the correct translational start codon in mAtpH
The results presented here indicate that Sthhiene-Dalgarno sequence present in the E. gracilis chloroplast atpH mRNA does play a positive role in enhancing theefficiency of translationalinitiation.The reduction intranslational efficiency observed upon mutation of the Shine-Dalgarno sequence is not as great as often observed with prokaryotic mRNAs but c c)

Summary

Introduction

A survey of higher plant chloroplast mRNA translational thestartcodon.Thechloroplast mRNA encoding the start sites has shown that over 90% have a sequence compleatpH gene has been selecteads an example oaf message mentary to thcehloroplast 16S rRNA sequence [7,8]. When mRNA prepared from this construct was tested for the ability to promote initiation complex formationwith chloroplast 30S subunits, about20-25% of the wild-type activity was observed (Fig. 3A).

Results

Conclusion