Isolation of a transcriptionally active chromosome from chloroplasts of Euglena gracilis

Abstract

A transcriptionally active chromosome was isolated in highly purified form from chloroplasts of E. gracilis. It contains chloroplast DNA, DNA-dependent RNA polymerase and other proteins. Transcription occurs at low levels of endogenous DNA, and is indifferent to high levels of exogenous DNA. RNA chain elongation continues for several h in vitro, and RNA chain initiation, determined by [.gamma.-32P]ATP incorporation, is continuous for at least 1 h in vitro. Maximal rates for RNA synthesis require only a divalent cation and the 4 ribonucleoside triphosphates. Apparent Km values for ATP, CTP, GTP and UTP are 4.0, 0.6, 2.5, and 2.3 .mu.M, respectively. As would be expected for a DNA-dependent RNA polymerase, RNA synthesis is inhibited by actinomycin D. However, rifampicin and streptolydigin, inhibitors of procaryotic RNA synthesis, and .alpha.-amanitin, an inhibitor of eucaryotic nuclear RNA polymerases II and III, do not inhibit the RNA synthesis reaction. Heparin, which is a potent inhibitor of the initiation of RNA synthesis by a nontemplate bound RNA polymerase, also does not inhibit RNA synthesis. Isolation of transcriptionally active chromosomes should prove to be a useful method to study the mechanism of selective RNA transcription of eucaryotic chromosomes.