Genome-wide promoter assembly in E. coli measured at single-base resolution

Abstract

When detected at single-base-pair resolution, the genome-wide location, occupancy level, and structural organization of DNA-binding proteins provide mechanistic insights into genome regulation. Here we use ChIP-exo to provide a near-base-pair resolution view of the epigenomic organization of the Escherichia coli transcription machinery and nucleoid structural proteins at the time when cells are growing exponentially and upon rapid reprogramming (acute heat shock). We examined the site specificity of three sigma factors (RpoD/σ70, RpoH/σ32, and RpoN/σ54), RNA polymerase (RNAP or RpoA, -B, -C), and two nucleoid proteins (Fis and IHF). We suggest that DNA shape at the flanks of cognate motifs helps drive site specificity. We find that although RNAP and sigma factors occupy active cognate promoters, RpoH and RpoN can occupy quiescent promoters without the presence of RNAP. Thus, promoter-bound sigma factors can be triggered to recruit RNAP by a mechanism that is distinct from an obligatory cycle of free sigma binding RNAP followed by promoter binding. These findings add new dimensions to how sigma factors achieve promoter specificity through DNA sequence and shape, and further define mechanistic steps in regulated genome-wide assembly of RNAP at promoters in E. coli.

Manipulation of topoisomerase expression inhibits cell division but not growth and reveals a distinctive promoter structure in Synechocystis

“…As an outlook, our strains should next be studied in diurnal conditions. A spatially resolved analysis of transcription along the genome ( 81 ), as well as DNA-structural footprinting methods, e.g., mapping of supercoiling-sensitive psoralen-binding sites ( 141 ), of gyrase-cleavage sites ( 21 ) or of the core transcription machinery ( 142 ) will provide an integrative picture of global regulatory mechanisms in a physiological context.…”

Asymmetry of both repressor and operator is important for the transcriptional regulation of theP. putidaXre-RES system

“…The basic HTH domain consists of three core helices making up a right-handed helical bundle with a partly open configuration, where typically two HTH domains are used to recognise the operator containing inverted or direct repeat sequence elements. The efficiency of binding of a repressor protein to an operator site depends on its binding affinity, which again is determined by base-specific readouts and intrinsic DNA shape, however, the exact mechanism by with repressor proteins specifically recognise their DNA binding site(s) in vivo remains unclear (7)(8)(9).…”