Abstract
Bacteria regulate gene expression to adapt to changing environments through transcriptional regulatory networks (TRNs). Although extensively studied, no TRN is fully characterized since the identity and activity of all the transcriptional regulators comprising a TRN are not known. Here, we experimentally evaluate 40 uncharacterized proteins in Escherichia coli K-12 MG1655, which were computationally predicted to be transcription factors (TFs). First, we used a multiplexed chromatin immunoprecipitation method combined with lambda exonuclease digestion (multiplexed ChIP-exo) assay to characterize binding sites for these candidate TFs; 34 of them were found to be DNA-binding proteins. We then compared the relative location between binding sites and RNA polymerase (RNAP). We found 48% (283/588) overlap between the TFs and RNAP. Finally, we used these data to infer potential functions for 10 of the 34 TFs with validated DNA binding sites and consensus binding motifs. Taken together, this study: (i) significantly expands the number of confirmed TFs to 276, close to the estimated total of about 280 TFs; (ii) provides putative functions for the newly discovered TFs and (iii) confirms the functions of four representative TFs through mutant phenotypes.
Highlights
Bacteria employ a broad range of mechanisms to regulate gene expression to achieve and maintain phenotypic states [1]
We present an overview of the binding sites determined by multiplexed chromatin immunoprecipitation (ChIP)-exo for these candidate transcription factors (TFs), highlighting their structural and functional properties
We describe the regulation of transcription initiation by these candidate TFs through a separate ChIP-exo screen for the RNA polymerase (RNAP) holoenzyme
Summary
Bacteria employ a broad range of mechanisms to regulate gene expression to achieve and maintain phenotypic states [1]. The primary mechanism by which gene expression is regulated in bacteria relies on the promoter recognition by the RNA polymerase (RNAP) holoenzyme and its subsequent initiation of transcription [2]. Since the core enzyme (including ␣, ␣, , ’ and ) itself is unable to recognize promoters or to initiate transcription, a sigma factor, which directly recognizes its target sequence, binds to the core enzyme, forming a complex known as the RNA polymerase holoenzyme. This complex orchestrates transcription initiation from specific promoters [1]. The identification of transcription factors and their association with sigma factors is fundamental to understanding how an organism responds to varying phenotypic demands through transcriptional regulation
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