The Mechanism of NusG-Mediated Transcription-Translation Coupling and The Role of RacR in Transcription Regulation in Escherichia coli

Transcription and translation are essential cellular processes that are coupled in bacteria. Though it was well-known that the rate of translation matches the rate of transcription, only in 2010 did evidence suggest direct physical coupling between the transcribing RNA polymerase (RNAP) and the translating ribosome. Nuclear magnetic resonance spectroscopy data showed that the RNAP-binding, transcription factor NusG could bind to the small ribosomal subunit protein, S10, through its C-terminal domain, thus, suggesting a model in which NusG simultaneously binds the transcription and translation machineries. In Chapter Two, I describe my investigations of the mechanism through which NusG-mediated transcription-translation coupling is established in bacteria, and how this coupling is regulated during gene expression. Specifically, I employed cell extract-based luciferase assays and purified C-terminal NusG mutants to show that the NusG N-terminal domain (NTD) and NusG F165A both inhibit transcription. This inhibitory effect was suppressed in an extract derived from a backtracking-resistant RNAP mutant strain, indicating that preventing backtracking by linking RNAP to the lead ribosome is a key function of NusG. While working with the cell extract-based luciferase assay system used to study NusG, I observed that deleting the cryptic rac prophage resulted in cell extracts with extremely low luciferase activity despite the strain having no phenotype in vivo. This initial observation grew into the project described in Chapter Three in which I explore the possibility of viral control of host genes by the poorly characterized rac prophage protein, RacR, through a combination of biochemical methods, structural modeling, bioinformatic analysis, and next-generation, transcriptome-wide, deep RNA sequencing. Taken together, the results reveal overlap between computationally predicted host gene targets and messenger RNA expression levels and suggest that RacR can function as a DNA-binding transcriptional regulator of host genes