show Abstracthide AbstractWhen performed at single bp resolution, the genome-wide location, occupancy level, and structural organization of DNA binding proteins provides mechanistic insights into genome regulation. Here we use ChIP-exo to provide the first high resolution view of the epigenomic organization of the E. coli transcription machinery and nucleoid structural proteins when cells are growing exponentially and upon rapid reprogramming (acute heat shock). We suggest that indirect readout of DNA shape at the flanks of cognate motifs provide major contributions to site specificity at promoter positions -35/-24 and -10/-12. We examined the site specificity of three sigma factors (RpoD/?70, RpoH/?32, and RpoN/?54), RNA polymerase (RNAP or RpoA, B, C) and two nucleoid proteins (Fis and IHF). Our results confirm and refine reports that RpoD binds most annotated promoters, whereas RpoH and RpoN bind a much smaller subset, each through their cognate motifs. However, only upon heat shock does RpoH becomes active for RNAP recruitment. In contrast, upon heat shock RpoD remains active at its cognate promoters (including at heat shock genes), whereas RpoN remains inactive at its cognate promoters. RpoN binds ~1,000 non-annotated RpoN motifs, which may reflect a large number of condition-specific transcription units. Occupancy patterns of sigma factors and RNAP suggest a common promoter recruitment mechanism that differs from the long-standing views that sigma and RNAP are co-recruited as a complex, and also simultaneously dissociate from promoters. Our findings suggest that sigma factors are recruited and/or maintained at most promoters via an RNAP-independent mechanism. When RNAP arrives, it dwells for a relatively short time before clearing the promoter, leaving sigma behind. Taken together these findings add new dimensions to how sigma factors achieve promoter specificity through DNA sequence and shape and redefine mechanistic steps in regulated promoter assembly in E. coli. Overall design: Genome-wide analysis of E. coli transcription factors using ChIP-exo