Enhanced translational utilization of chloroplast ribosomal protein mRNAs from two AUG codons shown by site-directed mutation

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Abstract
The cDNA for protein L12 of the chloroplast ribosome contains two ATG codons, both in consensus initiator context, as the beginning of its transit peptide coding region [Giese, K., and Subramanian, A. R. (1989) Biochemistry 28, 3525-3529]. Due to the location in the transit peptide, translational start from either ATG codon would yield the same mature protein after transport into chloroplasts and N-terminal cleavage. To test wheter this arrangement, also found in the cDNAs of several other chloroplast ribosomal proteins, could enhance the utilization of mRNA, we constructed a chimeric gene containing the 5'' part of the L12 cDNA fused to the neomycin phosphotransferase gene. A frameshift that would prematurely terminate the translation from the first AUG codon was introduced into the construct by site-directed mutagenesis. Transcription-translation of the fusion gene in vitro and its expression in vivo in spinach protoplasts showed that protein synthesis occurs from both initation codons: at 70-80% of the total level from the first and 20-30% from the second. The results thus show that (1) eukaryotic ribosomes can initiate to a significant level from a downstream AUG codon and (2) the occurrence of two in-frame initiation codons enhances translational efficiency.