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.

Fig. 1.

The tobacco chloroplast ndhC/K/J cluster and effects of 5′ deletions of ndhC/K mRNAs on ndhK translation. (A) Schematic of the ndhC/K/J cluster. Positions are relative to the ndhC AUG start codon (assigning A as “+1”). A bent arrow indicates the transcription start site (46). A partial mRNA sequence around the overlapping region is shown below. The ndhK start codon and the ndhC stop codon are in boldface type. Deduced amino acid sequences from ndhK and ndhC are shown above and below the mRNA sequence, respectively. Pathways 1 and 2 represent two translation routes. (B) Schematic of the test mRNA, in which most of the ndhK cistron was replaced with an mGFP-coding region (mgfp). Positions are as those on the ndh C/K/J cluster. Test mRNAs with progressive 5′ deletions are shown below the WT. (C) Gel patterns of translation products (mGFP fluorescence) separated by native PAGE after in vitro translation. (Right) Translation products from the test mRNAs for which the ndhC stop codon UAG was changed to UgG (mT1; Fig. 4A). The determined efficiencies of ndhK translation are shown as bar graphs below the gel pattern (WT value defined as 100%, n = 3, ± SEM). (D) Effects of stepwise 5′ deletions (7 nt each, indicated by arrows) from mRNA Δ153 (in B) on ndhK translation. Gel patterns of translation products are shown below the sequence. Note that a 7-nt deletion from the 5′ end (Δ160) still supported substantial ndhK translation.

Fig. 2.

Effect of internal deletions on ndhK translation by pathway 2. (A) Schematic of mRNA Δ153 (Fig. 1B). Test mRNAs with internal deletions (indicated as blanks, 39 nt each) are shown below. (B) Gel patterns of translation products as in Fig. 1C. Elements 1 and 2 are essential for ndhK translation by pathway 2. The determined efficiencies of ndhK translation are shown as bar graphs below (Δ153 value defined as 100%, n = 3, ± SEM).

Fig. 3.

Effects of mutations of internal AUG codons on ndhK translation by pathway 2. (A) Schematics of the wild type (wt) and Δ153 mRNAs (Figs. 1B and 2A). mRNA Δ153 was used to eliminate pathway 1 translation. The AUG at +1 represents the ndhC start codon. The AUGs at +151, +190, and +250 are internal in-frame AUGs within the ndhC cistron. Changes of AUGs to “uac” are shown below the Δ153. (B) Gel patterns of translation products from mRNAs with mutated AUGs as in Fig. 1C. (Lower) The determined efficiencies of ndhK translation are shown as bar graphs (Δ153 value defined as 100%, n = 3, ± SEM).

Fig. 4.

Effects of premature termination on ndhK translation by pathway 2. (A) Schematic of mRNA wt (Fig. 1B). AUGs are as in Fig. 3A. As a control, mT1 has a mutated UgG from the authentic ndhC stop codon UAG, which causes shifting of its stop codon (UAG) 22 codons farther downstream and, hence, no ndhK translation (Fig. 1C). mT2–mT6 possess mutations of internal codons to stop codons, with the first position indicated above (+118 to +265). Horizontal lines with arrowheads indicate translation proceeding up to stop codons. (B) Gel patterns of translation products from mRNAs with premature stop codons as in Fig. 1C. (Lower) The determined efficiencies of ndhK translation are shown as bar graphs [wild-type (wt) mRNA value defined as 100%, n = 3, ± SEM].

Fig. 5.

Detection of translation products from AUG₁₉₀. (A) Schematics of test mRNAs. mRNA Δ153 is in Fig. 2A. mRNA T7 has the T7 gene 10 5′UTR (68 nt) in front of AUG₁₉₀. (B) Gel patterns of translation products separated by denaturing PAGE. Translation reactions included fluorescently labeled lysyl-tRNA (left lanes) or fMet-tRNA (right lanes). Size markers were Coomassie-stained polypeptide standards (indicated at left) and FITC marker (center lane). (Lower) Magnified patterns around the 6.5-kDa regions.