cis- and trans-acting determinants for translation of psbD mRNA in Chlamydomonas reinhardtii

Chloroplast translation is mediated by nucleus-encoded factors that interac t with distinct cis-acting RNA elements. A U-rich sequence within the 5' un translated region of the psbD mRNA has previously been shown to be required for its translation in Chlamydomonas reinhardtii. By using UV cross-linkin g assays, we have identified a 40-kDa RNA binding protein, which binds to t he wild-type psbD leader, but is unable to recognize a nonfunctional leader mutant lacking the U-rich motif. RNA binding is restored in a chloroplast cis-acting suppressor,The functions of se,several site-directed psbD leader mutants were analyzed with transgenic C. reinhardtii chloroplasts and the in vitro RNA binding assay. A clear correlation between photosynthetic acti vity and the capability to bind RNA by the 40-kDa protein was observed, Fur thermore, the data obtained suggest that the poly(U) region serves as a mol ecular spacer between two previously characterized cis-acting elements, whi ch are involved in RNA stabilization and translation RNA-protein complex fo rmation depends on the nuclear Nac2 gene product that is part of a protein complex required for the stabilization of the psbD mRNA. The sedimentation properties of the 40-kDa RNA binding protein suggest that it interacts dire ctly with this Nac2 complex and, as a result, links processes of chloroplas t RNA metabolism and translation.