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This optional exercise deals with the problem of functional annotation for proteins derived from large-scale sequencing projects. The exercise is designed to illustrate both the usefulness and complexities in working with protein superfamilies. The lab assumes that you will return to earlier labs if you need help with a given web site that you have used before.
Lab 1 help guide Lab 2 help guide Lab 3 help guide
The gene ytfD in the B. subtilis genome is assigned two different putative functions associated with the accession numbers AAC00228 and F69991, respectively. But when ytfD was cloned and expressed in E. coli, it was found to have neither activity. The investigators who performed these experiments were, however, able to deduce the real function of ytfD from comparative genome analysis and show experimentally that ytfD does perform their predicted enzymatic function efficiently.
It was noted on the accession page for AAC00228 that this ytfD gene is "cotranscribed with menBE operon genes."
The menBE operon genes are associated with menaquinone biosynthesis in E. coli. In this organism, the pathway for menaquinone biosynthesis has been experimentally worked out and the role of each gene in the pathway has been verified.
Using this information, deduce the real function of ytfD in B. subtilis and justify your conclusion with multiple alignments/motif analysis.
Note: You can find an exposition of the genes of the menaquinone pathways in both E. coli and B. subtilis from the EcoCyc/MetaCyc database (http://ecocyc.org). As a start, you can use the "locations of mapped genes" link at the bottom of the menaquinone pathways page for E. coli to correlate the genes of this pathway with the reactions performed.
EBI's clustalw site ISREC's ProfileScan site NCBI's BLASTX site EcoCyc/MetaCyc database NCBI home page PDB site EXPASY's Prosite sites ( Canada, China, Korea, Taiwan, USA) NCBI's BLASTN site Pfam protein search site EXPASY's SIM Alignment sites ( Canada, China, Korea, Taiwan, USA) NCBI's BLASTP site