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Experimental determination of translational start sites resolves uncertainties in genomic open reading frame predictions - application to Mycobacterium tuberculosis

Smollett, KL; Fivian-Hughes, AS; Smith, JE; Chang, AC; Rao, T; Davis, EO; (2009) Experimental determination of translational start sites resolves uncertainties in genomic open reading frame predictions - application to Mycobacterium tuberculosis. MICROBIOL-SGM , 155 186 - 197. 10.1099/mic.0.022889-0.

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Abstract

Correct identification of translational start sites is important for understanding protein function and transcriptional regulation. The annotated translational start sites contained in genome databases are often predicted using bioinformatics and are rarely verified experimentally, and so are not all accurate. Therefore, we devised a simple approach for determining translational start sites using a combination of epitope tagging and frameshift mutagenesis. This assay was used to determine the start sites of three Mycobacterium tuberculosis proteins: LexA, SigC and Rv1955. We were able to show that proteins may begin before or after the predicted site. We also found that a small, non-annotated open reading frame upstream of Rv1955 was expressed as a protein, which we have designated Rv1954A. This approach is readily applicable to any bacterial species for which plasmid transformation can be achieved.

Type: Article
Title: Experimental determination of translational start sites resolves uncertainties in genomic open reading frame predictions - application to Mycobacterium tuberculosis
DOI: 10.1099/mic.0.022889-0
Keywords: OVERLAPPING GENES, INITIATION SITES, SIGMA FACTORS, EXPRESSION, SMEGMATIS, SEQUENCE, BACILLUS, MUTANTS, IDENTIFICATION, RECOGNITION
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology
URI: http://discovery.ucl.ac.uk/id/eprint/1329388
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