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

Abstract

Abstract When highly stable binary complexes of Escherichia coli RNA polymerase with T7 DNA are challenged with a mixture of rifampicin and the four ribonucleoside triphosphates, a major fraction of the enzyme molecules can initiate an RNA chain despite the presence of the drug. The exact fraction depends upon both the rifampicin concentration and the concentration of the ribonucleoside triphosphates: by raising the rifampicin concentration or lowering the ribonucleoside triphosphate concentration, the fraction of enzyme molecules inactivated by rifampicin progressively increases. Quantitatively, these results are most simply explained if RNA polymerase in a highly stable binary complex with T7 DNA is attacked by rifampicin at a constant rate in a second order reaction. The ability of enzyme molecules to initiate an RNA chain in such a challenge experiment is due to the relatively rapid rate of RNA chain initiation. There is no evidence that RNA polymerase in the highly stable binary complex can assume a conformation which is truly resistant to rifampicin prior to RNA chain initiation. This finding provides the basis for a specific assay for measuring the intrinsic rate of RNA chain initiation by E. coli RNA polymerase holoenzyme-T7 DNA binary complexes and for measuring the fraction of enzyme in such complexes which can initiate an RNA chain rapidly. The assay consists of varying the rifampicin concentration in a challenge experiment where both rifampicin and the ribonucleoside triphosphates are competing for RNA polymerase holoenzyme-T7 DNA complexes. At a given rifampicin concentration the half-life of a binary complex is limited to a known time interval, and consequently the intrinsic rate of RNA chain initiation can be measured. Under standard assay conditions, over 90 % of the holoenzyme molecules in binary complexes with T7 DNA initiate an RNA chain with an apparent first order rate constant for RNA chain initiation of 3.0 s-1 (t1/2 = 0.23 s). The fraction of enzyme molecules able to initiate an RNA chain rapidly (but not the rate of RNA chain initiation) depends on the amount of σ subunit in the enzyme preparation. Variation of the ribonucleoside triphosphate concentration in the RNA chain initiation assay, from 0.04 mm to 0.8 mm yields a linear relationship between the apparent first order rate constant for RNA chain initiation and the ribonucleoside triphosphate concentration.