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Microbiology 155 (2009), 186-197; DOI  10.1099/mic.0.022889-0IMMEDIATE OPEN ACCESS ARTICLE
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Microbiology 155 (2009), 186-197; DOI  10.1099/mic.0.022889-0
© 2009 Society for General Microbiology

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

Katherine L. Smollett{dagger}, Amanda S. Fivian-Hughes{dagger}, Joanne E. Smith, Anchi Chang, Tara Rao{ddagger} and Elaine O. Davis

Division of Mycobacterial Research, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK

Correspondence
Elaine O. Davis
edavis{at}nimr.mrc.ac.uk

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.

Abbreviations: ACDP, Advisory Committee on Dangerous Pathogens; RACE, rapid amplification of cDNA ends; SDM, site-directed mutagenesis

{dagger}These authors contributed equally to this work.

{ddagger}Present address: School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.






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