Translation of Overlapping Genes in Chloroplasts - a Novel Pathway to Translate the Downstream Cistron

The chloroplast NAD(P)H dehydrogenase complex consists of over 25 subunits, of which 11 are encoded by the tobacco chloroplast genome (ndhA-K). The ndhC and ndhK genes partially overlap and are cotranscribed in many plants. Using our in vitro translation system from tobacco chloroplasts, we showed that the tobacco ndhC/K genes are translationally coupled, but produce NdhC and NdhK in similar amounts. Generally, translation of the downstream cistron in overlapping mRNAs is very low because the majority of the ribosomes coming from upstream cistrons are released at the stop codon. Therefore, this finding suggested that the ndhK cistron is translated not only from the ndhC 5’UTR but also by an additional pathway. We then discovered that free ribosomes enter, with fMet-tRNA, at an internal AUG that is located in-frame in the middle of the upstream ndhC cistron, translate the 3’ half of the ndhC cistron and reach the ndhK start codon. Then, some of the ribosomes resume translation from the ndhK start codon. We detected a peptide corresponding to a 57 amino acid product encoded by the sequence from the internal AUG to the ndhC stop codon. We propose a model in which the internal initiation site AUG is not designed for synthesizing a functional isoform but for delivering additional ribosomes to the ndhK cistron to produce NdhK in the amount required for the assembly of the dehydrogenase. This pathway is a novel type of translation to produce protein in the needed amount with the cost of peptide synthesis.