Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.485131
Title: Structural investigation of proteins from the exosome superoperon of M. thermautotrophicus
Author: Leong, Ng Chyan
ISNI:       0000 0001 3608 2624
Awarding Body: University of York
Current Institution: University of York
Date of Award: 2007
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Abstract:
The exosome is a 3' to 5' RNA-processing and RNA-degrading macromolecular assembly. The genes encoding exosome core proteins are conserved in ar~haeal and eukaryotic cells. The work presented in this thesis is based on a structural and functional approach, using. protein crystallography, yeast two-hybrid experiments and EMSA assays, to study several proteins encoded in the exosOIne superoperon of Methanothermobacter thermautotrophicus (Mth). Crystal structures were determined for the gene products Mth680, Mth685, Mth689 and the exosome core, which consists of proteins encoded by the Mth682 and Mth683 genes. Research work was also carried out to identify protein interaction partners through protein-protein and protein-RNA interaction assays. The Mth680 protein is a member of the Imp4/Brix superfamily, which in eukaryotes is involved in ribosome biogenesis. We named this protein Mil for Mth Imp4.-Like. Its Xray structure provided the first information about the fold of proteins belonging to the Imp4/Brix superfamily. The structure revealed that the N-terminal and C-tenninal halves of this protein have very similar folds suggesting that the superfamily evolved by gene duplication. The fold of each half is similar to the fold of anticodon binding domain of class IIa aminoacyl-tRNA synthetases. A yeast-two hybrid experiment has identified the Mthl215 protein (homologue of the yeast Nopl protein) as a possible interaction partner of Mil. The binding of Mil to the exosome core has also been detected. Finally, Mil was also found to interact with single stranded RNA sequences. The Mth685 protein is an orthologue of the human Shwachman-Bodian-Diamond Syndrome protein (SBDS) therefore we named it mthSBDS. The structure of mthSBDS reveals a high degree of flexibility between the domains of this protein which are arranged in an 'L' shape. Several possible mthSBDS protein interaction partners including sensory transduction histidine kinase, alanyl tRNA synthetase: adenosylmethionine synthetase, Glutamate N-acetyltransferase and ribosomal proteins ofLl, L2, and Ll4 were identified. The function of Mth689 protein remains unknown. Its crystal structure reveals similarity to the large ribosomal subunit protein L5. Analysis of Mth689 structure reveals the presence of a positively charged concave surface that may interact with nucleic acids. I have also detefII}ined the X-ray structure of the Mth exosome core assembly that I prepared by co-expressing Mth682 and Mth683 proteins. The structure shows that the architechture of the active site and the positions of all active site residues are conserved, suggesting a similar phosphorolytic mechanism proposed for exosmes from other organisms. The structure shows for the fIrst time the presence of catalytically critical inorganic phosphate ions in each phosphorolytic active site further supporting the mechanism proposed earlier.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.485131  DOI: Not available
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