(4.1) The order in which GTFs bind at core promoters may be inferred from electr
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Question
(4.1) The order in which GTFs bind at core promoters may be inferred from electrophoretic mobility shift experiments: the binding of proteins to DNA molecules generally slows their speed of migration through a gel in an electric field. The autoradiograph of such a gel is shown here. 4- 2 N- 1 2 3 4 567 8 9 10 11 12 13 14 15 16 For experimental details see lecture 11 (i) Which of the four bands (labeled 1-4) above the naked DNA band (N) corresponds to which complex of DNA with general transcription factors and polymerase. Justify your conclusions. (This will require the comparison of two or more results (lanes). Hint: pay attention to lanes that correspond to reactions where individual factors were omitted.) (ii) In which order must factors bind? Justify your conclusion?Explanation / Answer
Band 1: represented by the naked DNA with D and A factors. As it is the only band showing when DNA bound with D and A.
Band 2: represented by the naked DNA binds with DAB proteins if any of them absent, the band will be absent.
Band 3: represented by the naked DNA binds with DAB proteins and RNApol along with F protein
Band 4: represented by the naked DNA binds with DAB RNA pol.
The order of protein binding must be
D-A-B-F-pol2
As in the absence of D there is no other protein binding to the DNA it must be the first protein to bind. B protein absence makes no F and Pol2 binding to DNA so it must be the next protein. In absence of F protein Pol 2 is not binding in all cases so it must be the succeeding to the F.
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