, Volume 269, Issue 3, pp 340-349
Date: 11 Apr 2003

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

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Abstract

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 Chlamydomonas in vivo. In this study, we have used chloroplast transformation and site-directed mutagenesis to analyse the functions of the 5′UTRs of tobacco psbA and rbcL fused to the coding region of the reporter gene uidA. Various mutant versions of the psbA leader, as well as rbcL/psbA hybrid leader elements, were investigated. Our results showed a 1.5- to 3-fold decrease in uidA mRNA levels and a 1.5- to 6-fold reduction in uidA translation efficiency in all psbA 5′UTR stem-loop mutants generated by sequence deletions and base alterations. This indicates that the correct primary sequence and secondary structure of the psbA 5′UTR stem-loop are required for mRNA stabilisation and translation. The 5′-terminal segment of the rbcL 5′UTR did not enhance the stability or translational activity of chimeric uidA mRNA 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.

Communicatred by R. G. Herrmann