Transcription is a crucial step in gene expression, orchestrated by RNA polymerase (RNAP), a molecular machine that transfers genetic information from DNA to RNA . Bacterial transcription provides a tractable model system which provides mechanistic insights on its more complex eukaryotic counterpart. Bacterial transcription is initiated after an RNAP holoenzyme (core RNAP bound to a σ initiation factor) melts the double-stranded DNA (dsDNA) around the transcription start to form a transcription bubble in the RNAP-promoter DNA open complex (RPo). Subsequently, RNAP performs cycles of RNA synthesis and dissociation (abortive initiation) and at a certain point, escapes from the promoter and enters elongation. RNAP has been studied extensively using genetic, biochemical and structural methods. Recent X-ray structures 3,4 vastly improved our understanding of transcription, leading to mechanistic proposals, and experiments that tested these proposals and further examined RNAP function. However, crystal structures cannot directly capture the dynamics of transcription. The transient nature, dynamics and heterogeneity of many intermediates hinders trapping and examination; it is often unclear whether a structure corresponds to an “on-pathway” intermediate or not. Analysis of intermediates is vital for understanding transcription, since it can reveal the order of steps during transcription and identify steps regulated by transcription factors and sequence elements (e.g., activators, repressors, DNA-bending proteins, promoter sequences). Analysis of intermediates also reports on conformational changes in DNA, RNAP, σ factors and activators during transcription.