34] Chloroplast p54 Endoribonuclease

Abstract

Much of the regulation of chloroplast gene expression has been assigned to stability determinants at the level of RNA precursors, including interactions of sequence and/or structural elements with their cognate binding proteins. A number of these proteins are known to assemble at the 5′-untranslated region (UTR) of chloroplast mRNAs, protecting them from exonucleolytic degradation and forming ribonucleoprotein (RNP) complexes involved in translational control. One of the proteins known to be controlled by phosphorylation is a 3′-endoribonuclease, which was first found to specifically bind to a conserved U-rich sequence element (UUUAUCU) of chloroplast precursor transcripts. This protein, which purifies as a monomeric polypeptide of an apparent molecular size of 54 kDa, was subsequently termed p54. This chapter presents the purification and properties of the p54 endoribonuclease from mustard (Sinapis alba), which might turn out to be a key player in posttranscriptional regulation of chloroplast gene expression. As a first step toward the purification of the p54 endonuclease, chloroplast protein extracts from mustard (S. alba) are prepared. Although the procedure has been developed for mustard, it has been found suitable for other higher plant species as well without significant modifications.