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

Abstract

The chloroplast NAD(P)H dehydrogenase ( NDH ) C ( ndhC ) and ndhK genes partially overlap and are cotranscribed in many plants. We previously reported that the tobacco ndhC/K genes are translationally coupled but produce NdhC and NdhK, subunits of the NDH complex, in similar amounts. Generally, translation of the downstream cistron in overlapping mRNAs is very low. Hence, these findings suggested that the ndhK cistron is translated not only from the ndhC 5′UTR but also by an additional pathway. Using an in vitro translation system from tobacco chloroplasts, we report here that free ribosomes enter, with formylmethionyl-tRNA fMet , at an internal AUG start codon that is located in frame in the middle of the upstream ndhC cistron, translate the 3′ half of the ndhC cistron, reach the ndhK start codon, and that, at that point, some ribosomes resume ndhK translation. 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 NDH complex. This pathway is a unique type of translation to produce protein in the needed amount with the cost of peptide synthesis.

Full transcription of the chloroplast genome in photosynthetic eukaryotes

“…Under such a polycistronic operon transcription model, plastome genes would be transcribed from intrinsic promoters and later form stable, size-fixed transcripts. However, this model cannot account for all the transcriptional products at whole-genome level, such as tremendous plastid noncoding RNA output 10 14 15 , pseudogene transcription 16 , multiple alternative promoters/terminators 17 18 , numerous heterogeneous and overlapping transcript isoforms 19 , and gene transcription uncoupling in the same polycistron 20 21 . These transcriptional dynamics and heterogeneity suggest that an additional general transcriptional mechanism triggers whole plastome transcription.…”

Seamless editing of the chloroplast genome in plants

“…Additional file 2 : Figure S2 shows the four overlapping gene pairs found in the tobacco cp genome: atpB-atpE , ndhC-ndhK , psbD-psbC and rpl22-rps3 gene [ 31 ]. The results from experiments using chloroplast lysates support translational coupling at psbD-psbC [ 37 ], ndhC-ndhK [ 38 , 56 ] but not atpB-atpE [ 57 ], which was consistent with ribosome profiling [ 58 ] but conflicts with an earlier study [ 59 ]; rpl22-rps3 remains untested. Analysis of bacterial genes found limited conservation of the sequences required for translational coupling [ 60 ].…”

“…The dependence of gusA expression on a complete upstream rbcL gene was consistent with translational coupling between the two genes. Translational coupling has been described in bacteria [ 35 , 36 , 53 ], bacteriophages [ 54 ], animal viruses [ 36 , 53 , 55 ] and appears to operate in chloroplasts, although it has only been demonstrated in vitro [ 37 , 38 , 56 ]. Additional file 2 : Figure S2 shows the four overlapping gene pairs found in the tobacco cp genome: atpB-atpE , ndhC-ndhK , psbD-psbC and rpl22-rps3 gene [ 31 ].…”

Translation in Mitochondria and Other Organelles

“…9.4a, the AUG start codon is located 4 nt upstream from the ndhC stop codon UAG. We proposed that the internal initiation site AUG is not designed for synthesizing a functional isoform but for delivering additional ribosomes (Yukawa and Sugiura 2013). Hence, the amount of ndhK product (NdhK) is expected to be low compared to NdhC.…”