Sequences downstream of the translation initiation codon are important determinants of translation efficiency in chloroplasts

The objective of this study was to determine if mRNA sequences downstream of the translation initiation codon are important for translation of plastid mRNAs. We have employed a transgenic approach, measuring accumulation of the neomycin phosphotransferase (NPTII) reporter enzyme translationally fused with 14 N-terminal amino acids encoded in the rbcL or atpB plastid genes. NPTII accumulation from wild-type and mutant rbcL and atpB segments was compared. We report that silent mutations in the rbcL segment reduced NPTII accumulation 35-fold. In contrast, mutations in the atpB mRNA reduced NPTII accumulation only moderately from approximately 7% (w/w) to approximately 4% (w/w) of the total soluble cellular protein, indicating that the importance of sequences downstream of the translation initiation codon are dependent on the individual mRNA. Information provided here will facilitate transgene design for high-level expression of recombinant proteins in chloroplasts by translational fusion with the N-terminal segment of highly expressed plastid genes or by introduction of silent mutations in the N-terminal part of the coding region.