Specific binding of chloroplast proteins in vitro to the 3' untranslated region of spinach chloroplast petD mRNA

Abstract

A detailed analysis of RNA-protein complex formation in the 3' untranslated region of spinach chloroplast petD mRNA has been carried out. Five chloroplast proteins that interact with petD RNA in this region, which contains an inverted repeat sequence capable of forming a hairpin structure, have been identified. A 33-kDa protein recognizes specifically the double-stranded stem of the hairpin structure; mutations that disrupt base pairing at the base of the stem reduce or eliminate protein binding. A 57-kDa protein recognizes specifically an AU-rich sequence motif that is highly conserved in petD genes of different higher plant species. The 57-kDa protein and possibly the 33-kDa protein form stable complexes with petD RNA in vitro and may interact with each other. In addition, their interaction with petD RNA is highly sensitive to heparin. The three other proteins, of 100, 32, and 28 kDa, display little sequence or structural binding specificity apart from their preference for uridine-rich sequences. They also interact with the 3' untranslated regions of other chloroplast RNAs such as those of psbA and rbcL. The functions of these proteins in the regulation of petD gene expression, including possible roles in transcription termination and RNA stability, are discussed.