Translational coupling of the maize chloroplast atpB and atpE genes

Abstract

The genes for the 13 and E subunits of maize chioroplast ATP synthase are encoded by the organelle genome, are cotranscribed, and have overlapping translation initiation and termination codons. To determine whether the atpB and atpE genes are translationally coupled, they were transformed into Escherichia coli on a multicopy plasmid. Synthesis of full-length 13 and E polypeptides demonstrated correct initiation of translation by the bacterial ribosomes. To assay for translational coupling, the promoter-distal alpE gene was fused to lacZ, resulting in the synthesis of an active hybrid B8-galactosidase. A frameshift mutation was introduced into the promoter-proximal atpB gene, and its effect on the transcription and translation of the atpE::lacZ fusion was measured. The mutation resulted in a 1000-to 2000-fold reduction in 13-galactosidase activity, but only a 2-fold decrease in LacZ mRNA synthesis rates or galactoside transacetylase levels. Similar results were obtained when the atpB/atpE::lacZ fusion and the atpB frameshift mutation were introduced into the photosynthetic cyanobacterium Synechocystis sp. PCC6803. We show that >99% of atpE translation depends on successful translation of atpB and, thus, conclude that the two genes are translationally coupled.The energy-transducing ATP synthase (ATPase) has a highly conserved structure that couples proton translocation across membranes with the synthesis of ATP. The complex present in chloroplasts, mitochondria, and prokaryotes consists of an F1, or CF1, extrinsic membrane component composed of five subunits (a-e) that catalyzes the final step in oxidative phosphorylation or photophosphorylation (1, 2). The integral membrane FO, or CFO, sector forms the proton channel. Genes for the a, /3, and 6 subunits of CF1 and subunits I, III, and IV of CFo are present in the chloroplast genome (3-6).The remaining three subunits (y,8, and II) are nuclear encoded and are imported into chloroplasts for assembly into an active enzyme. The chloroplast atp genes are organized into two transcriptional units that have retained a similar positional conservation to the arrangement ofgenes in the atp operon of Escherichia coli (3, 4). One cluster of genes has the order atpl/atpH/atpF/atpA, and there is evidence that they are cotranscribed (3,4). The other chloroplast-encoded atp genes are located many kilobases from the atpIHFA cluster (4-6), have the order atpB/atpE, and encode the /3 and E

A Rapid Ribosome Profiling Method Elucidates Chloroplast Ribosome Behavior in Vivo

“…For example, our results definitively show that translation of the maize atpB and atpE ORFs is not coupled in vivo, corroborating conclusions based on in vitro data (Suzuki et al, 2011) but differing from results obtained when these genes were expressed heterologously in bacteria (Gatenby et al, 1989). In addition, we were able to address for the first time whether splicing needs to precede translation initiation in chloroplasts.…”

“…The translation of overlapping ORFs in bacteria is typically coupled, in that translation of the second ORF is dependent on the translation of the first (reviewed in Jackson et al, 2007). However, there is conflicting data concerning the coupled translation of overlapping ORFs in chloroplasts: Analysis of the overlapping chloroplast atpB/atpE ORFs in heterologous expression systems pointed to coupled translation (Gatenby et al, 1989), whereas in vitro studies with a tobacco (Nicotiana tabacum) chloroplast translation extract suggested independent translation (Suzuki et al, 2011). (A) Follow-up to atp4 ribosome profile data suggesting a defect in rpl14 expression ( Figure 3C; see Supplemental Figure 2C online).…”

An upstream uncD sequence modulates translation of Escherichia coli uncC

“…This value is based on scans of lanes containing extracts of induced cells and subtraction of the background due to chromosomally encoded e (as in the lane corresponding to vector pUC8). In contrast to our results, Gatenby and co-workers (7) found that the production of chloroplast E is tightly coupled to translation of the gene encoding P. In that system, the stop codon for P overlaps the start codon for e. In E. coli, there are 20 bases, including a good Shine-Dalgarno sequence, between the two structural genes.…”

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

“…Translational coupling was also reported for the maize chloroplast atpB/E gene cluster, which has overlapping translation initiation and termination codons (AUGA), introduced into heterologous systems E. coli and Synecochocystis sp. PCC6803 (31). The chloroplast ATP synthase includes 3 ␤ subunits and 1 subunit, suggesting that translation efficiency of the downstream atpE cistron is lower than that of its upstream atpB cistron.…”