Nucleation of RNA chain formation by Escherichia coli DNA-dependent RNA polymerase

We have studied the early steps in RNA synthesis. The kinetic behaviour of the nascent RNA, having chain lengths between 3 and 11 bases, and the transcription fidelity were analysed using the bacteriophage T7 A1 promoter. By moving the stop-inducing base at position +12 in the wild-type template in single base steps upstream, a set of closely related templates was constructed which allowed stalling of the complexes in the registers 11, 10, 9 and 8. Using this set of templates sigma-factor release was determined. It occurs when RNA synthesis has proceeded to base position +9. Analysis of the RNA synthesis both with and without heparin yielded the following results: there are three kinds of complexes, (a) the well-known abortively transcribing complex, which is present until the RNA has reached a length of 5 bases, (b) an intermediate complex having RNA chain lengths between 6 and 8 bases, which is stably bound but has high forward as well as back reaction rates, (c) complexes with RNA chains consisting of more than 8 bases, which are stably bound and do not contain the sigma-factor. In general, the likelihood of chain elongation and the stability of the complexes increases with increasing RNA chain length in the early stages of RNA synthesis. Also the transcription fidelity increases correspondingly. Lack of fidelity leads to additional RNA products during the abortive state of transcription. "Read through" of RNA polymerase at stall positions of +8 to +11 also result from misincorporation.