Influence of the ionic environment on the in vitro transcription of the spinach plastid DNA by a selectively bound RNA-polymerase DNA complex

Abstract

The in vitro transcription of chloroplast DNA (ctDNA) is studied using a DNA-protein complex isolated from spinach plastids. The RNA products are compared to the in vivo synthesized ctRNA by competition for hybridization. At least 80% of the in vitro RNA sequences are present in vivo. Modifications of ionic strength or introduction of heparin in the reaction medium has an important effect on transcriptional activity of the complex. Furthermore, the length of the RNA chains increases ionic strength and amount of heparin. The RNA products are analysed by Southern hybridizations to EcoRI cTDNA fragments. Changes in the ionic strength or in the amount of heparin modify heterogeneously the transcription of the various DNA regions. The quantitative distribution of transcripts among the ctDNA fragments is used as evidence for the selectivity of the transcription. The activity of the DNA-protein complex is much more resistant to high ionic strength than an heterologous transcription system using Escherichia coli RNA polymerase and ctDNA. This latter system transcribes less ctDNA fragments.