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?
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?
