Termination codon-dependent translation of partially overlapping ndhC-ndhK transcripts in chloroplasts

Abstract

The chloroplast NAD(P)H dehydrogenase complex, a homologue of mitochondrial complex I, consists of >15 subunits, of which 11 are encoded by the chloroplast genome (ndhA-K). The ndhC and ndhK genes are partially overlapped and cotranscribed in many land plants. The downstream ndhK mRNA possesses 4 possible AUG initiation codons in many dicot plants. By using an efficient in vitro translation system from tobacco chloroplasts, we defined that the major initiation site of tobacco ndhK mRNAs is the third AUG that is located 4 nt upstream from the ndhC stop codon. Mutation of the ndhC stop codon (UAG) arrested translation of the ndhK cistron. Frameshift of the ndhC coding strand inhibited also translation of the distal cistron. The results indicated that ndhK translation depends on termination of the preceding cistron, namely translational coupling. Surprisingly, removal of the ndhC 5-UTR and its AUG still supported substantial translation of the ndhK cistron. This translation was abolished again by removing the ndhC stop codon. Although translation of the downstream cistron of an overlapping mRNA is generally very low, we found that the ndhC/K mRNA produces NdhK and NdhC in similar amounts. Based on subunit compositions of the bacterial complex I, the stoichiometry of NdhK and NdhC is suggested to be 1:1 in chloroplasts. To meet this stoichiometry, the ndhC/K mRNA is translated not only by a translational coupling event but also by a termination codondependent pathway.mRNA ͉ cistron ͉ initiation codon ͉ translational coupling

Additional pathway to translate the downstream ndhK cistron in partially overlapping ndhC-ndhK mRNAs in chloroplasts

“…Translational coupling requires a very short distance between stop and start codons, up to several nucleotides (27). When the distance increases, for example, by insertion of a premature stop codon in the upstream ndhC cistron, it abolishes translation of the downstream cistron (25). As shown in Fig.…”

“…Using our in vitro system, we previously reported that the initiation codon of the tobacco chloroplast ndhK cistron is located 4 nucleotides (nt) upstream from the ndhC stop codon (Fig. 1A) and that translation of the downstream ndhK cistron depends absolutely on translational termination of the upstream ndhC cistron, namely, translational coupling (25). This result can be explained by the downstream ndhK cistron being translated exclusively by the ribosomes that have completed translation of the ndhC cistron.…”

“…Translation products were separated by native polyacrylamide gel electrophoresis (PAGE) and monitored by mGFP fluorescence. This method allows detection of translation only from the ndhK start codon and has a very low background (25).…”

“…The observed fluctuation in translation efficiencies could be due to differences in secondary structures of the test mRNAs. We previously reported that the ndhC stop codon UAG is essential for ndhK translation (25). Stop codons UAA or UGA also act as UAG (Fig.…”

“…Based on the subunit composition of cyanobacterial NDH complexes (29,30), the amount of NdhK is likely to be similar to, or more than, that of NdhC. Our previous in vitro translation assay of the ndhC/K mRNA showed that the ndhK cistron is translated as efficiently, or slightly more efficiently, than the preceding ndhC cistron and that removal of the 5′ untranslated region (5′UTR) still supports substantial translation of the ndhK cistron (25). Therefore, in addition to translational coupling from the ndhC 5′UTR (pathway 1), the downstream ndhK cistron is likely translated by an additional mechanism (pathway 2), also in a termination-dependent manner (Fig.…”