33] Chloroplast mRNA 3′-end nuclease complex

Abstract

Publisher Summary This chapter discusses procedures for the purification of the proteins involved in chloroplast mRNA 3′-end processing, together with appropriate control assays to monitor the proteins throughout their purification. Control of mRNA stability has been establishedas an important mechanism for the regulation of chloroplast gene expression. The increase in RNA stabilization appears to be linked to specific processing events at the 5′ and 3′ ends of the respective mRNAs, but the exact mechanisms that link RNA processing and stability are still incompletely understood. The RNA in the 3′ untranslated region (3′ UTR) of most plastid mRNAs is predicted to fold into a stem–loop structure, which stabilizes the mRNA by protecting it from nucleolytic degradation. Several proteins involved in chloroplast mRNA 3′-end processing have now been identified, among them a high molecular weight protein (HMW) complex with an apparent molecular mass of approximately 550 kDa. Although the complete subunit structure of the complex remains to be elucidated, one of its components is a 100-kDa RNA-binding protein with an enzymatic activity that can degrade RNA processively in a 3′ → 5′ direction. The biochemical purification methods discussed in this chapter were developed to provide insights into the mechanisms and proteins involved in 3′-end processing, stabilization, and degradation of chloroplast RNAs.