Spt4/5 and its bacterial homologue NusG are the only known universally conserved RNAP- associated transcription elongation factors. In the hyperthermophilic archaeon Methanocaldococcus jannaschii, Spt5 comprises an N-terminal NGN domain and a C-terminal KOW domain, and is bound at its NGN domain by Spt4. NusG and Spt5 increase the processivity of RNAP by binding to the RNAP clamp via the NGN domain. This maintains the RNAP clamp in a closed conformation, thereby enabling RNAP to remain bound to the template DNA. The NusG KOW domain interacts with ribosomes, thereby coupling transcription to translation. The functions of Spt4/5 in archaea are less well characterised. The work contained within this thesis demonstrates that in the context of M. jannaschii cell extract, Spt4/5 is found in the same fractions as ribosomes and RNAP, and therefore has the potential to couple transcription and translation. Furthermore, data obtained by microscale thermophoresis suggests that the KOW domain of Spt5 interacts with purified ribosomes. Electron paramagnetic resonance was performed on Spt4/5, demonstrating that Spt5 is conformationally flexible, and that the presence of Spt4 restricts its mobility. Limited proteolysis and thermofluor assays support the notion that Spt4 stabilises the Spt5 NGN domain. In E. coli, NusA binds to RNAP as a component of the antitermination complex, along with NusG, NusB, and NusE. This enables RNAP to enter a pause and termination-resistant state. M. jannaschii NusA consists of two KH domains. Mutational analysis identified the contribution of the two KH domains to RNA binding and identified additional residues involved in the interaction. Archaeal NusA does not coelute with RNAP, raising the possibility that archaeal NusA does not have antitermination functions. In summary this thesis argues that Spt4/5 likely couples transcription and translation in archaea and indicates that archaeal NusA binds to RNA via a novel binding site.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:632106 |
| Date | January 2014 |
| Creators | Fielden, D. J. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1455740/ |
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