You are studying a gene (Gene X) in Escherichia coli that is expressed at 45 ºC

Question

You are studying a gene (Gene X) in Escherichia coli that is expressed at 45 ºC but not at 37 ºC. You have shown that the regulation of this gene occurs at the level of transcription. In addition, you have isolated a regulatory protein required for induction of transcription of gene X at 45 ºC and have demonstrated that the proteins binds to a specific 8-nucleotide sequence upstream (i.e. at the 5' flanking region) of the structural gene. You have cloned the complete gene X containing the upstream regulatory sequence in a plasmid, and you have available an E.coli mutant clone in which the entire gene X has been deleted. You now want to determine which of the nucleotide(s) in the 8-nucleotide sequence (a protein binding site) of gene X are involved in the interaction with the binding protein. How could you determine this experimentally?

Explanation / Answer

Given information,

Gene X is expressed only at 45'C but not at 35'C.

Its specific expression is induced at 45'C by a regulatory protein that binds to the upstream region of Gene X and activates it.

We know 8 nucleotide binding site of protein upstream to gene X.

We can do Electrophoretic mobility shift assay with the oligos of the binding sequence.

For example,

Assume that ATGCATGC is the 8 nucleotide sequence present upstream of gene X.

We can synthesize a primer contain this sequence and do an invitro binding assay. This can be done by end labelling the primer. So, if the protein binds to oligo, it will be retarded in the running gel.

Perform the same experiment by mutating single, double and triple/multiple nucleotides. In which mutation combinations the binding is inhibits, those residues are important for its binding. Once we know which sequences are important, we can clone WT as well as mutant sequences into a vector and transform E. coli.

Induce at 45'C and analyze whether the WT and mutant sequences can complement the gene X mutant.

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