Experimental determination of translational start sites resolves uncertainties in genomic open reading frame predictions - application to Mycobacterium tuberculosis.
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.
Smollett
Katherine L
KL
Division of Mycobacterial Research, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
Fivian-Hughes
Amanda S
AS
Division of Mycobacterial Research, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
Smith
Joanne E
JE
Division of Mycobacterial Research, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
Chang
Anchi
A
Division of Mycobacterial Research, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
Rao
Tara
T
Division of Mycobacterial Research, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
Davis
Elaine O
EO
Division of Mycobacterial Research, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D018387:Codon, Initiator; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000939:Epitopes; D016368:Frameshift Mutation; D016680:Genome, Bacterial; D006801:Humans; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000378:metabolism; D016366:Open Reading Frames / Q000235:genetics* / Q000502:physiology; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D018387:Codon, Initiator; D004268:DNA-Binding Proteins; D000939:Epitopes; D012697:Serine Endopeptidases; D012808:Sigma Factor; D000099160:LexA Repressor Protein; C081467:sigC protein, Bacteria