You have cloned four partial cDNAs that encode a transcription factor into a pla
ID: 53111 • Letter: Y
Question
You have cloned four partial cDNAs that encode a transcription factor into a plasmid that will generate a recombinant protein when expressed in bacteria. The partial cDNAs extend for different lengths towards the 5' end of the gene. Thus, you are able to express four proteins, each with the same C- terminus but with progressively longer stretches of amino acids in the N-terminus (see Map of the clones). Your goal is find the region of the transcription factor protein that binds to a particular DNA enhancer sequence. You express the cDNAs in bacteria, purify the four proteins and then mix the purified proteins with the enhancer DNA sequence that has been radioactively labeled and then analyze the mixtures on a native polyacrylamide gel (remember: native gels do not have SDS or mercaptoethanol and only radioactive DNA is visible on the gel). Your results are shown below (+ and – indicate which protein has been mixed with the DNA enhancer sequence).
A. Why are the shifted bands at different positions on the gel? (2 points)
Where is the binding domain for the enhancer DNA sequence located in the transcription factor? How can you tell? (3 points)
WhyweretherethreeshiftedbandswhentherecombinantproteinsfromcDNAclones3and4were mixed? What does this tell you about the structure of the transcription factor?
(3 points)
Explanation / Answer
A.
The results are not given.
The shift of the bands can be resulted due to binding of a protein complementarily to DNA at a specific sequence.
An enhancer is a short stretch of DNA of 1000 to 1500 base pairs to which proteins called transcription factors are bound and regulate the transcription and expression of the gene. Generally, the enhancer is located either upstream or downstream of the gene to which the transcription factor is to be bind.
Three shift bands of the recombinant proteins of CDNA clones 3 and 4 were mixed because the transcription factor might have subunits and or it may exist as a dimer.
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