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(American Journal of Botany. 2009;96:627-636.) doi: 10.3732/ajb.0800259 © 2009 Botanical Society of America, Inc.

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Efficient translation in chloroplasts requires element(s) upstream of the putative ribosome binding site from atpI¹

² Department of Biological Sciences, SUNY at Buffalo, Buffalo, New York 14260 USA ³ Department of Biochemistry and Molecular Biology, Colorado State University, Ft. Collins, Colorado 80523 USA

ABSTRACT

Thousands of proteins make up a chloroplast, but fewer than 100 are encoded by the chloroplast genome. Despite this low number, expression of chloroplast-encoded genes is essential for plant survival. Every chloroplast has its own gene expression system with a major regulatory point at the initiation of protein synthesis (translation). In chloroplasts, most protein-encoding genes contain elements resembling the ribosome binding sites (RBS) found in prokaryotes. In vitro, these putative chloroplast ribosome binding sequences vary in their ability to support translation. Here we report results from an investigation into effects of the predicted RBS for the tobacco chloroplast atpI gene on translation in vivo. Two reporter constructs, differing only in their 5'-untranslated regions (5'UTRs) were stably incorporated into tobacco chloroplast genomes and their expression analyzed. One 5'UTR was derived from the wild-type (WT) atpI gene. The second, Holo-substitution (Holo-sub), had nonchloroplast sequence replacing all wild-type nucleotides, except for the putative RBS. The abundance of reporter RNA was the same for both 5'UTRs. However, translation controlled by Holo-sub was less than 4% that controlled by WT. These in vivo experiments support the idea that translation initiation in land plant chloroplasts depends on 5'UTR elements outside the putative RBS.

Key Words: chloroplast • gene expression • land plant • ribosome binding site • translation • untranslated region

Received for publication 25 July 2008. Accepted for publication 21 October 2008.

FOOTNOTES

¹ The authors are deeply grateful to Dr. L. Allison, who collaborated in providing the initial chloroplast transformants and guided the transfer of chloroplast transformation technology to the Hollingsworth laboratory. They also thank Dr. M. Hanson and her colleagues for additional advice and training in chloroplast transformation and Dr. N. Ing for her careful reading and constructive suggestions on this manuscript. Support for this work was from a grant to M.J.H. from the Department of Energy (DE-FG02-00ER15103).

⁴ Author for correspondence (e-mail: hollings{at}buffalo.edu)

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