Substrate and cofactor requirements for RNA editing of chloroplast transcripts in Arabidopsis in vitro

Abstract

None of the macromolecular components of the chloroplast RNA editing apparatus has yet been identified. In order to facilitate biochemical purification and characterization of the chloroplast RNA editing apparatus, we have identified conditions suitable for production of chloroplast extracts from the model plant Arabidopsis that are capable of editing exogenous substrates produced by in vitro transcription. A simple poisoned primer extension assay readily quantified editing extent of mutated and wild-type substrates. Maximum editing efficiency typically varied from 10 to 40% with different chloroplast preparations. Substrates carrying as little as 47 nt surrounding the psbE editing site were as efficiently edited as longer substrates. Editing activity was stimulated when either ATP, CTP, or dCTP was provided to the extract, an unusual observation also recently seen with plant mitochondrial editing extracts. Editing was sensitive to a zinc chelator, also a characteristic of the mammalian APOBEC editing enzyme, which is a zinc-dependent cytidine deaminase.