Isolation and characterization of DNA-dependent RNA polymerase III from Leishmania mexicana and inhibition by purine analogs

Abstract

A DNA-dependent RNA polymerase has been isolated and characterized from the parasitic flagellated protozoan Leishmania mexicana. The Initial stages of purification utilized high-ionic-strength extraction and protamine sulfate treatment. The enzyme was further purified by differential elution by heparin-Sepharose, DEAE-Sephadex, and carboxymethyl-Sephadex chromatography. Analysis of the chromatographically purified RNA polymerase on nondenaturing gels revealed two electrophoretic forms. It has become increasingly important to understand the structure, function, and regulation of the eucaryotic RNA polymerases, since these enzymes may be necessary for specific transcriptional controls to be manifested in vitro as well as in vivo. A study of this enzyme in Leishmania species would not only lead to a better understanding of how some promising chemotherapeutic agents are incorporated into RNA or how they may inhibit protein synthesis, but might also provide information on the mechanism of promastigote-to-amastigote transformation. It is for these reasons that we have begun isolation and characterization of RNA polymerase in Leishmania mexicana.Nuclear extracts of eucaryotes can be fractionated by chromatography on DEAE-Sephadex to give three peaks of RNA polymerase activity. These three peaks correspond to three RNA polymerases (I, II, and III), which differ in relative amount, cellular location, type of RNA synthesized, subunit structure, response to salt and divalent-cation concentrations, and sensitivity to the mushroom-derived toxin ao-amanitin (6).