The catalytic agent in the transcription process is RNA polymerase. In E. coli, this enzyme consists of a core, which houses the fundamental transcription machinery, and a sigma factor (σ-factor), which guides the core to transcribe specific genes. The σ-factor facilitates the initiation of transcription by enabling the RNA polymerase holoenzyme to bind tightly to a promoter. This σ-dependent binding necessitates the localized melting of 10–17 base pairs of DNA near the transcription start site, forming an open promoter complex. By directing the holoenzyme to bind exclusively to certain promoters, the σ-factor determines which genes will be transcribed. Transcription initiation proceeds until 9 or 10 nucleotides are incorporated into the RNA, at which point the core transitions to an elongation-specific conformation, departs from the promoter, and continues with elongation. The σ-factor is generally released from the core polymerase, though not always immediately after promoter clearance, often exiting stochastically during elongation. The σ-factor can be reused by other core polymerases. Rifampicin sensitivity or resistance is governed by the core, not the σ-factor.

E. coli RNA polymerase achieves abortive transcription through a mechanism called scrunching, in which downstream DNA is drawn into the polymerase without the polymerase physically moving, while retaining its grip on the promoter DNA. The scrunched DNA may store sufficient energy to enable the polymerase to dissociate from the promoter and initiate productive transcription. Prokaryotic promoters contain two key regions located approximately 10 and 35 base pairs upstream of the transcription start site. In E. coli, these regions have consensus sequences of TATAAT and TTGACA, respectively. Generally, the closer a promoter's sequences match these consensus sequences, the stronger the promoter will be. Some exceptionally strong promoters also feature an additional element, known as an UP element, upstream of the core promoter.