Date of Award
Doctor of Philosophy (PhD)
Gene expression in bacteria is highly regulated at the step of transcription initiation. For decades, the vast majority of biochemical and kinetic studies have used Escherichia coli (Eco) RNA polymerase (RNAP) as a model for studying prokaryotic transcription initiation. However, properties of Eco RNAP are not representative of RNAPs from other bacterial species. Transcription in mycobacteria is distinct from Eco by its formation of unstable initiation complexes and its dependence on two transcription factors, RbpA and CarD, which are essential in the pathogen Mycobacteria tuberculosis (Mtb). In this thesis, I report the structural and biochemical characterization of RbpA and describe how it works synergistically with CarD to compensate for the unstable RNAPpromoter complexes formed in mycobacteria. I describe two crystal structures, Mtb RbpA-SID/σA 2 complex and RbpA bound to a Mycobacterium smegmatis (Msm) RNAP initiation complex, which reveal the structural mechanism of RbpA. The C-terminal σ- interacting domain (SID) of RbpA binds to domain 2 of σ (σA 2), while RbpA’s core domain interacts with the β’ subunit of RNAP. A basic linker (BL) connecting these two domains contacts promoter DNA upstream of the transcription bubble. Functional and kinetic analyses reveal that the interaction between the RbpA-BL and promoter DNA is the basis for transcription activation by RbpA. A synergistic effect between RbpA and CarD, striking in both transcription assays and in a kinetic assay measuring RPo formation, indicates that these two factors work together to activate transcription. By teasing apart the effects of the two factors on kinetic steps in RPo formation, this work shows that the RbpA and CarD cooperatively drive formation of a kinetic intermediate species during RNAP open complex formation. The work in this thesis provides a structural and functional understanding of transcription stimulation by RbpA and CarD, which we hypothesize make up part of the general transcription machinery in mycobacteria. There are currently no reported structures of mycobacterial RNAP, so the structure of the Msm RNAP initiation complex presented in this thesis will provide a platform for future studies studying the mycobacterial transcription system.
Hubin, Elizabeth, "Structural and Functional Studies of Mycobacterial General Transcription Factors RbpA and CarD" (2017). Student Theses and Dissertations. 407.