Chloroplast RNA polymerase from spinach: purification and DNA-binding proteins

Abstract

Spinach DNA dependent RNA polymerase was purified from isolated chloroplasts by two different procedures. Analysis of the protein composition of the two preparations by SDS-polyacrylamide gel electrophoresis always shows six abundant polypeptides with Mr of 150, 110, 102, 80, 75 and 38 Kd and one less abundant polypeptide of 25 Kd. Some other proteins ranging from 40-70 Kd in Mr are also detected but in a minor and variable amount. The two preparations have an optimum of enzyme activity at 30°C and at 15 mM (NH4)2SO4 when tested with denatured calf thymus DNA.Binding experiments with two different nick translated fragments of spinach chloroplast DNA show that the 80 and 75 Kd polypeptides possess a strong DNA binding capacity.