Date of Award
Doctor of Philosophy (PhD)
The σS subunit of RNA polymerase (RNAP) is the master regulator of stress responses in many Gram-negative bacteria. This alternative σ factor assembles with the core RNA polymerase to initiate the transcription of genes needed to survive different environmental changes. Crl is a small protein that activates the transcription of σSdependent genes. In contrast to most transcription activators, Crl does not bind DNA to help recruit RNA polymerase and instead interacts directly with σS. At the outset of my research, little was known about how the binding of Crl to σS leads to transcription activation. It was not clear if in addition to σS, Crl also made specific interactions with core RNAP. Using structural biology, molecular biology, biochemical and biophysical techniques, I gained novel insight into the unusual mechanism of Crl. This research validated and expanded on previous studies delineating the Crl/σS interaction and showed how a previously uncharacterized interaction between Crl and the β’ subunit of RNAP is critical for full transcription activation by Crl. This work advances our understanding of an unconventional mode of transcription activation in bacteria that might be more widespread than currently known. Chapter 1 provides background on bacterial transcription, σ factors, aspects of regulation, and closes with an introduction to Crl. Chapter 2 describes an approach that can be used to gain insight into the regulons of transcription factors like Crl. Most of the research in this thesis is presented in Chapter 3, which uses biochemical and biophysical approaches to elucidate how Crl activates transcription. Appendix A presents an attempt to study the surface of Crl that interacts with β’. Appendix B, briefly shows an attempt to investigate an additional mechanism by which Crl can activate transcription.
Cartagena, Alexis Jaramillo, "Structural and Functional Studies of the Unconventional Proteobacterial Transcription Activator CRL in Complex with the Transcription Machinery" (2020). Student Theses and Dissertations. 598.