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Molecular and Cellular Biology, December 1999, p. 8479-8491, Vol. 19, No. 12
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Small cis-Acting Sequences That Specify Secondary Structures in a Chloroplast mRNA Are Essential for RNA Stability and Translation

David C. Higgs,^1, Risa S. Shapiro,² Karen L. Kindle,^3, and David B. Stern^1,*

Boyce Thompson Institute for Plant Research,¹ and Division of Biological Sciences² and Plant Science Center,³ Cornell University, Ithaca, New York 14853

Nucleus-encoded proteins interact with cis-acting elements in chloroplast transcripts to promote RNA stability and translation. We have analyzed the structure and function of three such elements within the Chlamydomonas petD 5' untranslated region; petD encodes subunit IV of the cytochrome b₆/f complex. These elements were delineated by linker-scanning mutagenesis, and RNA secondary structures were investigated by mapping nuclease-sensitive sites in vitro and by in vivo dimethyl sulfate RNA modification. Element I spans a maximum of 8 nucleotides (nt) at the 5' end of the mRNA; it is essential for RNA stability and plays a role in translation. This element appears to form a small stem-loop that may interact with a previously described nucleus-encoded factor to block 5'3' exoribonucleolytic degradation. Elements II and III, located in the center and near the 3' end of the 5' untranslated region, respectively, are essential for translation, but mutations in these elements do not affect mRNA stability. Element II is a maximum of 16 nt in length, does not form an obvious secondary structure, and appears to bind proteins that protect it from dimethyl sulfate modification. Element III spans a maximum of 14 nt and appears to form a stem-loop in vivo, based on dimethyl sulfate modification and the sequences of intragenic suppressors of element III mutations. Furthermore, mutations in element II result in changes in the RNA structure near element III, consistent with a long-range interaction that may promote translation.

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^* Corresponding author. Mailing address: Boyce Thompson Institute for Plant Research, Cornell University, Tower Rd., Ithaca, NY 14853. Phone: (607) 254-1306. Fax: (607) 255-6695. E-mail: ds28{at}cornell.edu.

Present address: Department of Biological Sciences, University of WisconsinParkside, Kenosha, Wis.

Present address: Cereon Genomics, Cambridge, Mass.

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Molecular and Cellular Biology, December 1999, p. 8479-8491, Vol. 19, No. 12
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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