CRYSTAL STRUCTURE AND BIOCHEMICAL ANALYSIS OF ARCHAEAL RNA POLYMERASE TRANSCRIPTION ELONGATION FACTORS
Open Access
- Author:
- Klein, Brianna Joy
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- September 07, 2010
- Committee Members:
- Katsuhiko Murakami, Dissertation Advisor/Co-Advisor
Katsuhiko Murakami, Committee Chair/Co-Chair
Paul Lee Babitzke, Committee Member
David Scott Gilmour, Committee Member
B Tracy Nixon, Committee Member
Biao He, Committee Member - Keywords:
- RNA-directed RNA synthesis by DNA-dependent aRNAP
Pyrococcus furiosus Spt4/5
Structure of Spt4/5 at 1.8 Å
Archaeal RNAP
Inhibited TECs With or Without TFS - Abstract:
- ABSTRACT Transcription factors regulate each of the stages of transcription: initiation, elongation, and termination. Throughout my dissertation I focused on three directions of elongation. The first, Spt4/5 in archaea, eukaryotes, and bacteria (NusG), is the only conserved elongation factor in all three domains of life. Here, I report the X-ray crystal structure of the Pyrococcus furiosus Spt4/5 complex solved at 1.8 Å resolution. This is the first complete Spt4/5 X-ray crystal structure containing Spt4 (zinc and N-terminal NusG (NGN) binding domains) and the Spt5 N-terminal, linker and C-terminal Kyprides-Ouzounis-Woese (KOW) domains. Spt4 contacts Spt5 through the linker, KOW, and NGN. Thus, Spt4 may restrict the location of the KOW with respect to the Spt5NGN domain, suggesting the Spt5 linker between NGN and KOW is rigid. With our collaborators we solved a cryo-EM reconstruction of Spt4/5-archaeal RNAP (aRNAP) and engineered a model of the Spt4/5-aRNAP elongation complex providing new features about Spt4/5. The second direction is RNA synthesis, occurs during transcription elongation, is not foolproof and incorrect bases can be introduced. Removal of mismatches occurs by a proofreading mechanism. I demonstrate that aRNAP is capable of intrinsic and TFS-enhanced RNA cleavage of a transcription elongation complex (TEC) assembled on a DNA/RNA scaffold. Altering reaction conditions, it is possible to inhibit RNA cleavage and capture steps of the cleavage mechanism for crystallization, to determine the X-ray crystal structures of the proofreading TEC in the presence and absence of TFS. The final direction focuses on hepatitis delta virus (HDV), an RNA satellite virus of the hepatitis B virus that infects about 20 million people worldwide. HDV has a single-stranded circular RNA genome without its own RNA-dependent RNAP. Instead, HDV replication and mRNA synthesis is carried out by host RNAP without DNA intermediates. Using aRNAP and a short model RNA template containing a hairpin loop and dinucleotide bulge, I determined the aRNAP forms a stable complex with the model RNA. In the presence of Mg2+ aRNAP cleaves the RNA around the dinucleotide bulge and carries out RNA extension. This shows how a DNA-dependent aRNAP cleaves and synthesizes RNA, extending the known abilities of aRNAP.