Molecular and functional properties of highly purified transcriptionally active chromosomes from spinach chloroplasts

In the present work we describe the preparation of highly purified, transcriptionally active chromosomes (TACs) from spinach chloroplasts by refinement and improvement of the method commonly used so far. The plastid DNA-bound TAC-RNA polymerase and soluble plastid RNA polymerase were efficiently separated from each other with a single gel filtration step that is preceded by protamine sulfate precipitation and treatment with heparin. Removal of all detergents from the extraction buffer in a further purification step drastically increases the specific transcriptional activity and yields a 23000-fold purification compared to the crude chloroplast extract. The purified TAC extract reveals 10 highly enriched proteins among 30-40 identifiable polypeptides. The rpoA gene product encoded by the plastid DNA was copurified with the transcriptional activity through all purification steps, confirming its functional integrity in the TAC complex. The transcript patterns of crude and highly purified TAC extracts are identical for 10 selected chloroplast genes, indicating no influence of the purification steps on transcriptional selectivity. The reported method provides a basis: for the identification and analysis of the components of the TAC fraction in general and of the TAG-associated RNA polymerase in particular.