Studies of Ribonucleic Acid Chain Initiation by Escherichia coli Ribonucleic Acid Polymerase Bound to T7 Deoxyribonucleic Acid

When highly stable binary complexes are formed between Escherichia coli RNA polymerase and T7 DNA at low molar ratios of enzyme to DNA (fewer than three enzyme molecules per DNA) a major fraction of the enzyme molecules is able to initiate an RNA chain extremely rapidly (t1/2 ∼ 0.2 s) when presented with the four ribonucleoside triphosphates. The initiation of T7 RNA chains by binary complexes is altered in two ways when elevated concentrations of KCl are included in the reaction. For binary complexes formed in the absence of KCl, the presence of 0.2 m KCl during initiation lowers the rate of chain initiation about 5-fold but does not affect the fraction of enzyme molecules which can initiate an RNA chain rapidly. However when binary complexes are formed in the presence of increasing amounts of KCl, the fraction of enzyme molecules which can initiate an RNA chain is progressively decreased, even when initiation takes place after dilution to lower salt concentrations. Evidence is presented that binary complexes formed in the presence of KCl contain enzyme bound at or near the T7 early promoter region but that the enzyme is in a state (I complex) which is unable to initiate an RNA chain or can initiate only very slowly. The enzyme in the I complex is in equilibrium with enzyme in a state (RS complex) which can rapidly initiate T7 RNA chains.