Gene transcription is a fundamental cellular process carried out by RNA polymerase (RNAP) enzymes and is highly regulated through the action of gene regulatory complexes. Important mechanistic insights have been gained from structural studies on multisubunit RNAP from bacteria, yeast and archaea, although the initiation process that involves the conversion of the inactive transcription complex to an active one has yet to be fully understood. RNAPs are unambiguously closely related in structure and function across all kingdoms of life and have conserved mechanisms. In bacteria, sigma (sigma) factors direct RNAP to specific promoter sites and the RNAP/sigma holoenzyme can either form a stable closed complex that is incompetent for transcription (as in the case of sigma(54)) or can spontaneously proceed to an open complex that is competent for transcription (as in the case of sigma(70)). The conversion of the RNAP/sigma(54) closed complex to an open complex requires ATP hydrolysis by enhancer-binding proteins, hence providing an ideal model system for studying the initiation process biochemically and structurally. In this review, we present recent structural studies of the two major bacterial RNAP holoenzymes and focus on mechanistic advances in the transcription initiation process via enhancer-binding proteins.