Studies of DNA‐dependent RNA polymerase

DNA‐dependent RNA polymerase reacts with a nucleic acid to form a binary complex, which can be isolated by density gradient centrifugation or by millipore filtration techniques. Studies of the complex of Escherichia coli RNA polymerase and phage lambda DNA show that temperature and ribonucloside triphosphates do not affect the extent of binding. The binary complex is very sensitive to ionic strength. Enzyme bound in the complex exchanges with free enzyme. In the presence of Mg 2+ , the lambda DNA becomes saturated with enzyme when 2.5 μg have been bound per microgram of DNA. Transfer RNA and synthetic polyribonucleotides also react with the enzyme at 0°C. In the presence of divalent cation at low ionic strength, the main complex of the enzyme and a polyribonucleotide has a sedimentation value of 15S. In the absence of divalent cation, or at higher ionic strength, the value is 13S. Polyuridylate and polycytidylate have a greater affinity for the enzyme than polyadenylate or transfer RNA. An interesting question regarding the specificity of interaction of RNA polymerase with native DNA is whether any of the RNA chains made have the sequence AUG, the principal N ‐formylmethionine codon, at the 5′ termini. Studies with T7 DNA show that, under conditions of high ionic strength, 24% of the RNA chains start August.