A role for initiation codon context in chloroplast translation.
To study the role of initiation codon context in chloroplast protein synthesis, we mutated the three nucleotides immediately upstream of the initiation codon (the -1 triplet) of two chloroplast genes in the alga Chlamydomonas reinhardtii. In prokaryotes, the -1 triplet has been proposed to base pair with either the 530 loop of 16S rRNA or the extended anticodon of fMet-tRNA. We found that in vivo, none of the chloroplast mutations affected mRNA stability. However, certain mutations did cause a temperature-sensitive decrease in translation and a more dramatic decrease at room temperature when combined with an AUU initiation codon. These mutations disrupt the proposed extended base pairing interaction with the fMet-tRNA anticodon loop, suggesting that this interaction may be important in vivo. Mutations that would still permit base pairing with the 530 loop of the 16S rRNA also had a negative effect on translation, suggesting that this interaction does not occur in vivo. Extended base pairing surrounding the initiation codon may be part of a mechanism to compensate for the lack of a classic Shine-Dalgarno rRNA interaction in the translation of some chloroplast mRNAs.
Esposito
D
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Department of Molecular Biology and Genetics and Boyce Thompson Institute for Plant Research, Cornell University, Tower Road, Ithaca, New York 14853, USA.
Hicks
A J
AJ
Stern
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DB
D000818:Animals; D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D002736:Chloroplasts / Q000235:genetics*; D003062:Codon / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D010442:Peptide Chain Initiation, Translational; D010940:Plant Proteins / Q000235:genetics; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D014170:Transformation, Genetic
D003062:Codon; D010940:Plant Proteins; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S