Structural Basis of Bacterial Transcription: Investigation of Mechanisms of Reiterative Transcription and Ribosomal RNA Transcription by X-ray Crystallography and Cryo-Electron Microscopy
Open Access
- Author:
- Shin, Yeonoh
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 21, 2020
- Committee Members:
- Katsuhiko Murakami, Dissertation Advisor/Co-Advisor
Katsuhiko Murakami, Committee Chair/Co-Chair
Paul Babitzke, Committee Member
David Scott Gilmour, Committee Member
Philip C Bevilacqua, Outside Member
Lu Bai, Committee Member
Wendy Hanna-Rose, Program Head/Chair - Keywords:
- Transcription
RNA polymerase
X-ray crystallography
Cryo-electron microscopy
Ribosomal RNA
Reiterative transcription - Abstract:
- Visualizing the three-dimensional structure of a macromolecule in a biological system provides many new insights into the function and mechanism of the biological process. This can be achieved by solving the atomic resolution structures of macromolecules by X-ray crystallography and cryo-electron microscopy (cryo-EM). I investigated the mechanism of two different types of bacterial transcription systems by structural biology approaches. The first type is conditional reiterative transcription which regulates gene expression by nucleoside triphosphate substrate availability. Using time-dependent soak-trigger-freeze X-ray crystallography, I determined the transcript initiation and reiterative transcription complex structures from the Bacillus subtilis pyrG promoter that show 1) the unique pathway of RNA extension; and 2) a mismatch between template DNA and the incoming nucleotide followed by base sharing with an upstream template DNA during the reiterative RNA transcription. I also determined the structures of transcript initiation complexes from the Escherichia coli pyrBI promoter and B. subtilis pyrG promoter variants that show an alternative mechanism of RNA slippage and extension pathway compared with the pyrG promoter. Ribosomal RNA (rRNA) is the highest expressed gene in rapidly growing bacteria; however, it is drastically downregulated by the DksA/ppGpp binding to RNAP during stationary phase and stressed conditions. I investigated the mechanism of extraordinary active rRNA transcription and its DksA/ppGpp-dependent inhibition by cryo-EM structure determination of RNAP and rRNA promoter complex and biochemical assays. The comparison of the apo-form of RNAP, closed and open complex structures, and DNA opening assay reveals the role of σ non-conserved region (σNCR) rotation in facilitating DNA unwinding. The structures also show the structural basis of RNAP-UP element binding and the “open complex scrunching” by which the template DNA contacts with σ finger and β' lid and transcript start site selection at the rRNA promoter. DksA/ppGpp binding to RNAP induces the movement of RNAP mobile domains that influence the stability of the open complex and hinder the DNA unwinding of promoters containing GC-rich discriminator sequence. I propose that promoter-dependent pleotropic effect of DksA/ppGpp is based on the duplex stability in discriminator sequences.