A role for initiation codon context in chloroplast translation

To study the role of initiation codon context in chloroplast protein synthe sis, we mutated the three nucleotides immediately upstream of the initiatio n 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-tRN A. We found that in vivo, none of the chloroplast mutations affected mRNA s tability. However, certain mutations did cause a temperature-sensitive decr ease in translation and a more dramatic decrease at room temperature when c ombined with an AUU initiation codon. These mutations disrupt the proposed extended base pairing interaction with the fMet-tRNA anticodon loop, sugges ting that this interaction may be important in vivo. Mutations that would s till permit base pairing with the 530 loop of the 16S rRNA also had a negat ive 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.