- Title
- Increased understanding of the molecular interactions involved in bacterial transcription and recombination
- Creator
- Keller, Andrew N.
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2014
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Transcription is the transfer of genetic information from genomic DNA into RNA, and it is performed in bacteria by RNA polymerase (RNAP). The subunit composition of RNAP generally consists of five core subunits, α2ββ’ω; however, this can vary in some organisms. Furthermore, the activity of RNAP is dependent on an entourage of transcription factors, which interact directly or indirectly with RNAP to modulate its activity. The Bacillus subtilis YkzG protein has traditionally been misrepresented as a second ω-subunit due to its small size and tight association with RNAP. This work determined the YkzG structure using X-ray crystallography and identified that it has structural similarity to the λ-phage Gp2 protein. It also showed that YkzG-deficient cells exhibited wild-type transcript levels and that despite the genes for YkzG and the ribonuclease Rnase J1 being transcriptionally and translationally linked in a highly expressed operon, they are unlikely to be functionally related. Using targeted mutagenesis, this work suggests that YkzG binds to B. subtilis RNAP at the β’-jaw domain using an equivalent mechanism to Gp2. Work was also performed that examined the interaction between the essential transcription factor NusA and RNAP. NusA N-terminal domain (NusAN) constructs were purified for Nuclear Magnetic Resonance experiments and crystallisation screening. While no NusA structures were determined in this work, it provided an excellent foundation for the structural characterisation of the NusA-RNAP interaction. The Xer site-specific recombination pathway is used by bacteria to separate chromosomes that during replication, have become dimerised or catenated. Central to this recombination reaction by the heterodimer formed between XerC and XerD, is their activation at midcell through a direct interaction between XerD and the cell division protein FtsK. Using X-ray crystallography, the structure was determined of truncated XerD and FtsK constructs in complex. Consequently, the interaction interface between XerD and FtsK was identified and the structure was used to construct a model for the activation of Xer site-specific recombination. This work proposes that FtsK activates recombination by stabilising the interaction between XerC and XerD across a synaptic complex.
- Subject
- transrciption; DNA; RNA; RNAP
- Identifier
- http://hdl.handle.net/1959.13/1055293
- Identifier
- uon:15863
- Rights
- Copyright 2014 Andrew N. Keller
- Language
- eng
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| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details Download | ATTACHMENT01 | Abstract | 161 KB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Thesis | 28 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT03 | Appendices | 343 KB | Adobe Acrobat PDF | View Details Download |