Abstract

Chloroplasts carry out photosynthesis in plant cells and algae, and possess their own genomes. Nuclear encoded gene products synthesized in the cytosol are transported into the chloroplast to control the gene expression. In the green algae Chlamydomonas reinhardtii , translation of the psbC mRNA, which encodes subunit P6 of the photosystem II, has been shown to be regulated by the TBC1, TBC2 and TBC3 nuclear products via its 547 nt long 5 ' untranslated region (5 ' UTR). The middle region (222-320) is absolutely required for translation and is predicted to form a stable stem-loop structure. The chloroplast mutant psbC-FuD34 modifies the sequence of this stem-loop and abolishes the translation. It is predicted to increase the stem-loop stability. Another chloroplast mutation, psbC-F34suI , partially suppresses the requirement for the TBC1 factor and is predicted to decrease the 222-320 stem-loop stability. This study did not reveal a precursor 5 ' end that needs to be processed in psbC transcripts, contrary to other mRNAs encoding PSII subunits. Dimethyl sulfate was used in vivo and in vitro to analyse 5 ' UTR structures. It did not clearly establish the presence of the 222-320 stem-loop structure, but revealed some structural differences between the wild-type and psbC-FuD34 . The in vivo methylation pattern generally agreed with computer predicted secondary structure. Temperature gradient gel electrophoresis was used to compare the melting profiles of wild type and several mutant 5 ' UTRs. Important differences in the unfolding pattern exist between the wild type, psbC-FuD34 and deletion mutants near the middle stem-loop. Moreover, more than one conformation can exist at the same conditions. This study suggests that structural interactions with the 222-320 stem-loop play an important role in the translation of the psbC mRNA.