Genome-scale analyses of transcription and transcriptional regulation in bacteria

The textbook model of bacterial transcription regulation posits that promoters occur immediately upstream of genes and that transcription factors (TFs) modulate transcription through promoter-proximal binding. However, the recent application of unbiased genome-wide approaches, such as ChIP-seq and RNA-seq, has revealed a much more complex picture, including TF binding and transcription initiation occurring in unexpected locations. This dissertation describes the use of deep sequencing-based approaches to evaluate the genome-wide binding of transcription-related proteins and identify locations of transcription initiation. I have assessed the genome-wide binding of three Escherichia coli TFs and an alternative sigma factor. Additionally, I have analyzed genome-wide patterns of transcription initiation in both E. coli and Salmonella enterica. Two of the TFs evaluated fit the textbook model of transcription regulation, with binding limited to promoter-proximal regions. However, one TF and the alternative sigma factor bound many sites inside genes and far from gene starts. These intragenic binding events showed no evidence of regulatory function or conservation, and may instead represent spurious binding or biological noise. Analysis of genome-wide transcription start sites in E. coli and S. enterica provided additional evidence of intragenic transcription initiation. A small, but highly significant number of intragenic transcription start sites were conserved between E. coli and S. enterica, suggesting they correspond to functional RNAs. These conserved intragenic RNAs are ideal candidates for future functional characterization. The phenomena of intragenic TF binding and intragenic transcription initiation are still poorly understood, but are prevalent across all domains of life. While many intragenic binding and/or initiation events likely represent biological noise, there is evidence, such as the limited conservation detected in this study, that a subset of these events are functionally important. Additional work is necessary to understand (i) why some TFs only bind in promoter-proximal regions while others bind more diffusely, (ii) to identify which intragenic TF binding sites, promoters, and RNAs are functional and which are not, and (iii) to understand the potential functions of these non-canonical binding sites, promoters, and RNAs.