The conserved secondary structural RNA motifs of EncephaloMyoCarditis Virus (EMCV) have been well characterised biochemically and shown to play an important role in translation initiation by a novel cap-independent mechanism called Internal Ribosomal Entry Site (IRES). However, the three dimensional structure and interactions of these conserved motifs are not known, and hence the mechanism is not fully understood. The NMR results described in this thesis have provided, for the first time, new structural knowledge on the conformation of these motifs, their affinity for Mg2+ and their intermolecular interactions. RNA motifs selected from two separate domains (I and J) of the IRES structure were investigated using a range of 2D and 3D NMR techniques. The apical ‘hammerhead’ region of the I domain contains a highly conserved 16mer RNA which hosts a stable and mutationally sensitive G547CGA550 tetraloop. Sequence specific assignments were carried out on this motif, along with its Mg2+ complex, and a large number of NMR experimental constraints were generated for the RNA structure determination. Similarly, high resolution NMR structures of a distal 17mer RNA, which has been predicted to be a potential receptor for the GCGA tetraloop, and its Mg2+ complex were also produced. Thus, we were able to demonstrate that Mg2+ stabilises the RNA tertiary structure via non-specific interactions. Since the largest changes were induced at the tetraloop motif, we propose that Mg2+ stabilises the 16mer into an optimum conformation which is essential for IRES function. The determination of the structures of the above motifs led us to investigate the 16mer-17mer binary (1:1) complex at 1 GHz, in the presence of Mg2+. Significant changes were observed in the 1H and 31P chemical shift, NOE intensity and line width, clearly demonstrating RNA-RNA interactions taking place between the two components. The most interesting result to emerge was the distinct absence of NOEs from G547{NH} of the stable tetraloop, thus highlighting an important structural role for this functionally critical residue. Since no previous work has shown a clear interaction between the two RNAs, the results obtained in this project provide the first direct experimental evidence for intramolecular interactions in the I domain of EMCV IRES.Finally, we show how isotopically labelled RNAs can be successfully used as an aid in NMR assignment, analysis and structure determination. The J domain of EMCV IRES binds to eIF4GII protein and is essential for translation initiation. A suite of 3D NMR techniques were carried out on a highly enriched and uniformly 13C, 15N-labelled 39mer RNA. Several key features of the RNA, which may be involved in protein recognition, were identified. Further, a selectively 19F-labelled 16mer RNA from the I domain, was also studied to show how fluorine NMR can be used to probe RNA structure, dynamics and interactions. The RNA motifs of the EMCV IRES were shown to exhibit high stabilities, which are brought about by the complex folding of the various secondary structural elements involving RNA- Mg2+, RNA-RNA and RNA-protein tertiary interactions. It is these vital interactions that enable the IRES to recruit the ribosome in the translation initiation step of protein synthesis, and have laid a strong foundation for further NMR investigation of the whole IRES.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:554168 |
| Date | January 2012 |
| Creators | Mohammed, Sadia |
| Contributors | Ramesh, Vasudevan |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/nmr-studies-of-the-structure-dynamics-and-interactions-of-the-conserved-rna-motifs-of-the-emcv-picornavirus(2cc0fa5b-f80d-48f2-a918-44b7bf5a2429).html |
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