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Title: Nuclear translation
Author: Baboo, Sabyasachi
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2012
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In bacteria, protein synthesis can occur tightly coupled to transcription. In eukaryotes, it is believed that translation occurs solely in the cytoplasm; I test whether some occurs in nuclei and find: (1) L-azidohomoalanine (Aha) – a methionine analogue (detected by microscopy after attaching a fluorescent tag using ‘click’ chemistry) – is incorporated within 5 s into nuclei in a process sensitive to the translation inhibitor, anisomycin. (2) Puromycin – another inhibitor that end-labels nascent peptides (detected by immuno-fluorescence) – is similarly incorporated in a manner sensitive to a transcriptional inhibitor. (3) CD2 – a non-nuclear protein – is found in nuclei close to the nascent RNA that encodes it (detected by combining indirect immuno-labelling with RNA fluorescence in situ hybridization using intronic probes); faulty (nascent) RNA is destroyed by a quality-control mechanism sensitive to translational inhibitors. I conclude that substantial translation occurs in the nucleus, with some being closely coupled to transcription and the associated proof-reading. Moreover, most peptides made in both the nucleus and cytoplasm are degraded soon after they are made with half-lives of about one minute. I also collaborated on two additional projects: the purification of mega-complexes (transcription ‘factories’) containing RNA polymerases I, II, or III (I used immuno-fluorescence to confirm that each contained the expected constituents), and the demonstration that some ‘factories’ specialize in transcribing genes responding to tumour necrosis factor α – a cytokine that signals through NFκB (I used RNA fluorescence in situ hybridization coupled with immuno-labelling to show active NFκB is found in factories transcribing responsive genes).
Supervisor: Cook, Peter Richard Sponsor: Felix Scholarship
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biology ; Cell Biology ; Microscopy ; Protein chemistry ; Synthetic organic chemistry ; Nuclear translation ; nuclear protein synthesis ; transcription factory ; nonsense-mediated mRNA decay ; puromycin ; L-azidohomoalanine ; click chemistry ; Copper-catalyzed azide-alkyne cycloaddition ; intronic translation ; immuno-RNA FISH ; proximity ligation assay ; antibody blocking