Stability determinants in the chloroplast psbB/T/H mRNAs of Chlamydomonas reinhardtii

Abstract

The chloroplast gene psbB encodes the chlorophyll-a binding protein P5 (CP47), one of the core subunits of photosystem II (PSII). The psbB mRNA and the downstream psbT and psbH transcripts fail to accumulate in the Chlamydomonas reinhardtii nuclear mutant 222E affected in the Mbb1 gene (Monod et al. 1992, Mol. Gen. Genet. 231, 449-459). By introducing chimeric genes consisting of sequences from psbB and the reporter gene aadA into the chloroplast, the target site of Mbb1 was mapped in the psbB 5' untranslated region (UTR). Primer extension analysis indicates that the psbB RNA exists in a less abundant long form and a more abundant short form, with 5' ends at positions -147 and -35 relative to the AUG initiation codon, respectively. The longer transcript is present both in the wild type (WT) and 222E mutant, but the shorter one accumulates only in the WT. Two putative stem-loop structures in the longer 5' UTR can be deleted individually without affecting psbB mRNA accumulation. Insertion of a poly G cassette in the long leader stabilizes a chimeric psbB transcript in the 222E mutant, suggesting the involvement of a 5'-3' exonuclease. We also show that psbH and psbT are transcribed from the upstream psbB gene promoter, and that the psbH mRNA has its own target sequence for Mbb1 function. We discuss the role of this nucleus-encoded factor, required for specific chloroplast gene expression, in the assembly of the multi-protein PSII complex.