Translational coupling of the maize chloroplast atpB and atpE genes

Translational coupling of the maize chloroplast atpB and atpE genes

^*Institute for Enzyme Research, University of Wisconsin, Madison, WI 53706
^†E. I. du Pont de Nemours and Company, Wilmington, DE 19880
^§Plant Breeding Institute, Trumpington, Cambridge CB2 2LQ, United Kingdom

Abstract

The genes for the β and ε subunits of maize chloroplast 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 β and ε polypeptides demonstrated correct initiation of translation by the bacterial ribosomes. To assay for translational coupling, the promoter-distal atpE gene was fused to lacZ, resulting in the synthesis of an active hybrid β-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 β-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.

Footnotes

↵ ‡ To whom reprint requests should be addressed at: E. I. du Pont de Nemours and Company, Experimental Station, P.O. Box 80402, Wilmington, DE 19880-0402.
↵ ¶ Present address: Department of Molecular Biology and Genetics, University of Guelph, Guelph, ON, N1G 2W1, Canada.
↵ ǁ Present address: Department of Biological Chemistry, University of California, Irvine, CA 92717.