Mutations that alter the higher-order structure of its 5' untranslated region affect the stability of chloroplast rps7 m
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O R I GI N A L P A P E R
D. C. Fargo á E. Hu á J. E. Boynton á N. W. Gillham
Mutations that alter the higher-order structure of its 5¢ untranslated region affect the stability of chloroplast rps7 mRNA
Received: 3 May 2000 / Accepted: 4 July 2000 / Published online: 23 August 2000 Ó Springer-Verlag 2000
Abstract In this paper, we examine the eects of mutations in the 5¢UTR of the chloroplast rps7 transcript of Chlamydomonas reinhardtii that reduce the stability of the mRNA. Five point mutants in the rps7 5¢UTR were selected on the basis of their failure to accumulate reporter mRNA in Escherichia coli. Each of these mutations produces alterations in the predicted higher-order structures of the rps7 5¢UTR that destabilize the mRNA. Cis-acting suppressors of these mutations have been selected in E. coli and in the C. reinhardtii chloroplast that restore message stability and function. No dierences in RNA melting and reannealing pro®les have been observed between wild type, original mutant, and suppressor 5¢UTRs transcribed in vitro. Proteins of 32 kDa and 47 kDa that bind to the wild-type rps7 5¢UTR are not detected by UV cross-linking assays performed with any of the mutant rps7 5¢UTRs. However, binding of the 32-kDa protein is restored in the six suppressor mutants examined. This suggests that the 32-kDa protein may be involved in protecting the rps7 5¢UTR and the attached coding region from digestion by ribonucleases. Alternatively, the binding site for the 32-kDa protein may be independently lost in the rearranged tertiary structure of the mutant 5¢UTR that exposes the RNA to degradation and is restored in the suppressor mutants. Key words Chlamydomonas á Chloroplast á RNA stability á 5¢UTR
Communicated by R. G. Herrmann D. C. Fargo á E. Hu á J. E. Boynton (&) á N. W. Gillham Developmental, Cell and Molecular Biology Group, Departments, of Botany and Zoology, Box 91000, Duke University LSRC, Building, Research Drive, Durham NC 27708, USA E-mail: [email protected]
Introduction Despite sharing numerous characteristics with its prokaryotic relatives, the translational apparatus of the chloroplast exhibits several important dierences with respect to the well-de®ned Escherichia coli model (Gold 1988; Zengel and Lindahl 1994; Blattner et al. 1997). These include both the mechanisms that control initiation of translation and the overall structure of individual mRNAs. Major post-transcriptional dierences relative to E. coli are the greater in vivo stability (half-life) of chloroplast mRNAs, the increase in median size of the chloroplast 5¢ untranslated regions (UTRs), and the absence of coupling of chloroplast transcription and translation (reviewed in Stern and Drager 1998). While the majority of analyses of mRNA stability in chloroplasts have focused on processing in the 3¢UTR (Nickelsen 1998), this study examines the signi®cance of speci®c 5¢UTR sequences for mRNA stability. Primary sequences of chloroplast 5¢UTRs exhibit little conservation, although their predicted secondary structures are highly folded
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