In vivo analysis of plastid psbA, rbcL and rpl32 UTR elements by chloroplast transformation: tobacco plastid gene expression is controlled by modulation of transcript levels and translation efficiency.
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abstract
5' and 3' untranslated regions (UTRs) of plastid RNAs act as regulatory elements for post-transcriptional control of gene expression. Polyethylene glycol-mediated plastid transformation with UTR-GUS reporter gene fusions was used to study the function of the psbA, rbcL and rpl32 UTRs in vivo. All gene fusions were expressed from the same promoter, i.e. the promoter of the 16S-rRNA gene, such that variations in RNA and protein levels would be due to the involved UTR elements alone. Transgenic tobacco lines containing different combinations of UTRs showed fivefold variation in the uidA-mRNA level (RNA stability) and approximately 100-fold differences in GUS activity, a measure of translation activity. The rbcL 5'-UTR conferred greater mRNA stability than the psbA 5'-UTR on uidA transcripts. In contrast, the psbA 5'-UTR enhanced translation of GUS to a much greater extent compared to the rbcL 5'-UTR. The psbA 5'-UTR also mediated light-induced activation of translation which was not observed with other constructs. Deletion mutagenesis of an unanalysed terminal sequence element of the psbA 5'-UTR resulted in a twofold drop in uidA-mRNA level and a fourfold decrease in translation efficiency. Exchange of 3'-UTRs results in up to fivefold changes of mRNA levels and does not significantly influence translation efficiency. The mechanical impacts of these results on plastid translation regulation are discussed.
