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).
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:595905 |
| Date | January 2012 |
| Creators | Baboo, Sabyasachi |
| Contributors | Cook, Peter Richard |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:5266f049-d576-44fd-ab26-11cf7a27f678 |
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