Redefining the Coordination of Gene Expression Machineries in Bacillus subtilis

Abstract

Transcription-translation coupling has long been considered a defining feature of bacterial gene expression. As soon as the ribosome binding site emerges from the RNAP, the ribosome can initiate translation, and both physically associate and kinetically coordinate with the RNAP over the course of transcription. This close proximity between the RNAP and ribosome allows the ribosome to modulate the fate of the transcribing RNAP, and forms the basis of many regulatory mechanisms, including translation-mediated transcription attenuation and transcriptional polarity. However, transcription-translation coupling has remained largely uncharacterized outside of Escherichia coli and other closely related bacteria. In this thesis, I describe an alternative mode of gene expression, runaway transcription, utilized by the bacterium Bacillus subtilis. Through measurement of transcription and translation kinetics, I demonstrate that the RNAP outpaces the ribosome in B. subtilis, resulting in large distance between the RNAP and ribosome that sets alternative rules for gene expression in this bacterium. In particular, runaway transcription helps to explain the increased use of riboswitches and RNA binding proteins in regulating transcription attenuation. In addition, I show that runaway transcription necessitates a more limited role for Rho-dependent transcription termination in B. subtilis, whereby Rho activity is selectively targeted to specific regions of the genome by cis-encoded sequence elements. Subsequent characterization of these Rho termination events across conditions reveals the Rho termination on specific transcripts can be regulated, providing an additional physiological role for this protein. Together, this characterization of runaway transcription and its consequences in B. subtilis provides insights into the underlying principles that have shaped the evolution of divergent regulatory mechanisms across bacteria.