1. ATP synthase is a well conserved multimeric protein. All organisms studied to
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Question
1. ATP synthase is a well conserved multimeric protein. All organisms studied to date contain homologs of 8 different subunits which are essential for the complexes function. However, you have just completed sequencing the genome of a novel organism, which despite having a functional ATP synthase, it only has genes which are homologous to 5 of the 8 essential subunits. On the basis of these findings you hypothesise that the ATP synthase of the novel organism contains novel subunits which fulfil the same functional role as the missing essential subunits. Describe, a series of experiments you could perform to identify the novel subunits and the genes that code for them.Explanation / Answer
Dry lab approach :
Multiple sequence alignment of the subunit sequences to find the conserved region(conserved region might essentially be the active site region of the enzyme)
If sequence homology is found,find out if the more than one active site sequence is now included in the novel organisms genome.(one squence having the coding region of more than one subunit hence same gene for 2 or more different subunits)....this way genes can be maped onto the chromosomes.
Wet lab approach:
Isolate the protein.....run SDS-PAGE ,every subunit sample can be loaded by using proteases or a mixture of proteases.See the difference of the subunit lengths and infer the size of the subunits based on the ladder used.
If any of the subunit has a lesser mobility on the gel that means the function of a certain subunit has been compensated by this subunit.
Isolate the protein fragment from the gel and perform western blotting.Mass spec can be done to predict the peptide sequence and hence the corresponding DNA/RNA sequence can be known.Perform GISH with the formed primers to locate the DNA sequence that will give the position of the gene in the genome.Repeate the same for other fragments on the gel.
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