The downstream atpE cistron is efficiently translated via its own cis-element in partially overlapping atpB-atpE dicistronic mRNAs in chloroplasts

Abstract

The chloroplast atpB and atpE genes encode subunits β and ε of the ATP synthase, respectively. They are co-transcribed as dicistronic mRNAs in flowering plants. An unusual feature is an overlap (AUGA) of the atpB stop codon (UGA) with the atpE start codon (AUG). Hence, atpE translation has been believed to depend on atpB translation (i.e. translational coupling). Using an in vitro translation system from tobacco chloroplasts, we showed that both atpB and atpE cistrons are translated from the tobacco dicistronic mRNA, and that the efficiency of atpB translation is higher than that of atpE translation. When the atpB 5'-UTR was replaced with lower efficiency 5'-UTRs, atpE translation was higher than atpB translation. Removal of the entire atpB 5'-UTR arrested atpB translation but atpE translation still proceeded. Introduction of a premature stop codon in the atpB cistron did not abolish atpE translation. These results indicate that atpE translation is independent of atpB translation. Mutation analysis showed that the atpE cistron possesses its own cis-element(s) for translation, located ~25 nt upstream from the start codon.

Figure 1.

The tobacco chloroplast atpB/atpE operon and its transcripts. Nucleotide positions relative to AUG start codons (A as +1) are shown above for atpB and below for atpE. Bent arrows indicate atpB and atpE transcription start sites (TSSs) (31,42–44). A vertical arrow indicates a processing site (−90) (42). The common 3′-end is designated by closed triangles (31). AUG, atpB start codon; AUGA, overlapping atpB stop-atpE start codons. A partial mRNA sequence around the atpE start codon is shown below. Deduced amino acid sequences from atpB and atpE genes (β- and ε-subunits) are shown above and below the mRNA sequence, respectively.

Figure 2.

Translation of the atpE cistron from dicistronic and monocistronic mRNAs. (A) mRNA templates. A large portion of the atpE cistron was replaced by the Citrine-coding region (citrine). Positions are as in Figure 1. The full sequence is in Supplementary Figure S3. (B) Gel patterns of translation products. Synthesized Citrine products were detected by 488-nm light (left panel) and 532-nm light (right panel). Lanes labeled C, no-mRNA template control. Di and Mono indicate dicistronic and monocistronic mRNAs, respectively. Citrine bands represent atpE products (ε-subunit). Faint bands above the main Citrine bands are probably non-specific fluorescent substances in the S30 fraction.

Figure 3.

Effect of atpB translation on atpE translation. (A) mRNA templates with atpB and atpE cistrons replaced by EGFP (egfp) and Citrine (citrine)-coding regions, respectively. Positions are as in Figure 1. The full sequence is in Supplementary Figure S5. (B) Gel patterns of translation products. Lanes labeled C, no-mRNA control. The 5′-UTR was either from atpB, atpH or rbcL, and lane ‘–‘ denotes no 5′-UTR or following AUG (Supplementary Figure S5). Synthesized EGFP and Citrine products were detected by 488-nm light (left panel) and 532-nm light (right panel). EGFP and Citrine bands represent atpB and atpE products (β- and ε-subunits), respectively. Quantification of translation products based on intensity of fluorescent bands (atpB value defined as 100%) is shown in bar graphs below. Faint bands above the main EGFP bands could be loosely folded EGFP.

Figure 4.

Effect of premature termination of the upstream atpB cistron on atpE translation. (A) mRNA templates as in Figures 3A (Di) and 2A (Mono). Bold arrows indicate locations of premature termination codons. The last U (position −69) of UAU was replaced with A to create a stop codon (UAA) in the 3′ atpB cistron or in the atpE 5′-UTR. (B) Gel patterns of translation products. Lanes labeled C, no-mRNA control. Di and Mono indicate dicistronic and monocistronic mRNAs, respectively. ‘wt’, wild-type (UAU); ‘mut’, mutant (UAG). Synthesized EGFP and Citrine products were detected as in Figure 3. The EGFP product from the wt mRNA includes a β subunit fragment (27 amino acids) at the C-terminus. The premature termination deleted 24 amino acids from this extension, and therefore the band migrated faster. Faint bands above the main EGFP bands could be loosely folded EGFP.

Figure 5.

Effect of a frameshift mutation in the atpB cistron on atpE translation. (A) mRNA templates (as in Figure 2A) and location of the frameshift. Insertion of 4 nt at the first BglII site caused a frameshift, creating a stop codon within the atpB cistron. ‘wt’, wild type; ‘mut’, frameshift mutant. Sequences around the frameshift are in Supplementary Figure S2. (B) Gel patterns of translation products. Lanes labeled C, no-mRNA control. Synthesized Citrine products were detected by 532-nm light.

Figure 6.

Effect of internal deletions in the atpE 5′-UTR on atpE translation. (A) The mRNA (as in Figure 3A) and partial atpE 5′-UTR sequence. The SD-like sequence and the overlapping stop and start codons are underlined. mRNAs with internal deletions (indicated as blanks, from −80 to −3) are shown below. Terminal sequences of egfp and citrine are boxed. The gcu triplet after +36 is a ligation product. (B) Gel patterns of translation products. Synthesized EGFP and Citrine products were detected by 488-nm light (upper panel) and 532-nm light (lower panel). White arrowheads in the upper panel point to EGFP bands and black arrowheads in the lower panel point to Citrine bands. In the upper panel, migration of EGFP bands differed due to charge and size differences on native gels; i.e. deletion of one negatively charged glutamate (E⁻) caused slower migration (‘−23’ to ‘-20’ and ‘−17’ to ‘−14’) and deletion of positively charged lysine (K⁺) caused faster migration (‘−5’ to ‘−2’).

Figure 7.

Effect of mutations in the atpE 5′-UTR on atpE translation. (A) mRNA templates as in Figures 3A (Dicistronic) and 2A (Monocistronic) and partial atpE 5′-UTR sequence. The SD-like sequence and the overlapping stop and start codons are underlined. Nucleotide and amino acid sequences are in Supplementary Figure S8. (B) Gel patterns of translation products. Lanes labeled C, no-mRNA control. Synthesized EGFP and Citrine products were detected as in Figure 6. In the upper left panel, migration of EGFP bands differed due to charge differences on native gels. The m1 and m3 dense bands overlap Citrine and EGFP products. Quantification of Citrine products based on intensity of fluorescent bands (wt value defined as 100%) is shown in bar graphs below.