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.
Publication types
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Evaluation Study
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Bacterial Proteins / chemistry
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Bacterial Proteins / genetics*
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Bacterial Proteins / metabolism
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Base Sequence
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Codon, Initiator*
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DNA-Binding Proteins / chemistry
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / metabolism
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Epitopes
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Frameshift Mutation
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Genome, Bacterial
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Humans
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Molecular Sequence Data
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Mycobacterium tuberculosis / genetics*
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Mycobacterium tuberculosis / metabolism
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Open Reading Frames / genetics*
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Open Reading Frames / physiology
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Plasmids / genetics
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Protein Biosynthesis*
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Serine Endopeptidases / chemistry
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Serine Endopeptidases / genetics
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Serine Endopeptidases / metabolism
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Sigma Factor / chemistry
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Sigma Factor / genetics
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Sigma Factor / metabolism
Substances
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Bacterial Proteins
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Codon, Initiator
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DNA-Binding Proteins
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Epitopes
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LexA protein, Bacteria
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Sigma Factor
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sigC protein, Bacteria
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Serine Endopeptidases