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.
Highlights
Mapping the protein translational start site for genes is essential to define the protein sequence, intergenic distances and upstream DNA regions which may contain regulatory motifs
E. coli was grown in Luria–Bertani (LB) broth or on LB agar plates, while M. smegmatis and M. tuberculosis were grown in modified Dubos medium (Difco) supplemented with 4 % albumin and 0.2 % (w/v) glycerol or on Difco Middlebrook 7H11 agar (Beckton Dickinson) plates supplemented with 4 % albumin and 0.5 % (w/v) glycerol
E. coli and M. smegmatis liquid cultures were grown at 37 uC with shaking at 225 r.p.m. and M. tuberculosis was grown at 37 uC in a rolling incubator at 2 r.p.m
Summary
Mapping the protein translational start site for genes is essential to define the protein sequence, intergenic distances and upstream DNA regions which may contain regulatory motifs. Correct identification of translational start sites is necessary for understanding both protein function and transcriptional regulation (Makita et al, 2007; Rison et al, 2007). One of the most widely used for prokaryotic genomes is GLIMMER, which tends to use the first possible translation initiation codon (ATG, GTG or TTG) for a particular gene, giving the longest possible ORF (Delcher et al, 1999). Other approaches take into account factors such as the location of ribosome-binding sites and protein sequence comparisons (Besemer et al, 2001; Makita et al, 2007; Nielsen & Krogh, 2005).
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