The stem-loop region of the tobacco psbA 5'UTR is an important determinant of mRNA stability and translation efficiency

Regulation of chloroplast gene expression involves networked and concerted interactions of nucleus-encoded factors with their target sites on untranslated regions (UTRs)of chloroplast transcripts. So far, only a few cis-acting elements within such 5'UTR sequences have been identified as functional determinants of mRNA stability and efficient translation in Chlamydomonasin vivo. In this study, we have used chloroplast transformation and site-directed mutagenesis to analyse the functions of the 5'UTRs of tobacco psbAand rbcLfused to the coding region of the reporter gene uidA. Various mutant versions of the psbAleader, as well as rbcL/psbAhybrid leader elements, were investigated. Our results showed a 1.5- to 3-fold decrease in uidAmRNA levels and a 1.5- to 6-fold reduction in uidAtranslation efficiency in all psbA5'UTR stem-loop mutants generated by sequence deletions and base alterations. This indicates that the correct primary sequence and secondary structure of the psbA5'UTR stem-loop are required for mRNA stabilisation and translation. The 5'-terminal segment of the rbcL5'UTR did not enhance the stability or translational activity of chimeric uidAmRNA under the standard light-dark regime of 16 h light and 8 h dark. Stabilising effects were, however, observed when the cells were kept continuously in the dark. Possible reasons for the influence of the 5'UTR of the tobacco psbA on mRNA stability and translation efficiency are discussed.