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nline net/bbcswebdav pid 2729738-dt-content-rid-20518143-1/courses/B2740-1552/Practice%20Exam%202%282%29 pdf 2) Your friend decides to place Green Fluorescent Protein (GFP) under the control of the promoter from the lacZ gene we discussed in class. She put this expression plasmid into a bacterium. The promoter from the lacZ gene is diagrammed to the right. start site for RNA synthesls site a) Recall that two important gene regulatory proteins that bind to this promoter are the lac repressor and the CAP protein. Your friend grows the bacteria with the expression plasmid on media (food) with no glucose and no lactose and found that the sleep-induced protein could not be purified from the bacteria. Is this surprising to you, given how the lacZ promoter works? Explain how the binding of the gene regulatory factors lead to this situation. (5 points) b) How would you recommend your friend grow her bacteria so that she will get maximal expression of GFP when using the lacZ promoter? (3 points) You decide to express GFP in a fly instead ofa bacterium. The fly promoter has overlapping binding sites for Krüppel and Bicoid so that only one of the two proteins can bind to DNA at any one time (in other words, Krüppel and Bicoid cannot bind simultaneously). You conduct some experiments using a cell free transcriptional system, where you add the expression plasmid and equal quantities of Krüppel and Bicoid proteins and get the results in the Table 1 to the left protein added GFP mRNA produced? none no Bicoid Krüppel and Bicoid no c) According to this data, which protein(s) is likely a gene activator? (2 points)Explanation / Answer
2.a. It is not surprising. The regulatory factor lac repressor (that comes from the gene lacl) doesn´t allowed the transcription to ocurr, unless the lactose is added. A metabolite from the lactose will inactivate it, this metabolite is called allolactose. This repressor binds to a short sequence of DNA and doesn´t allow the binding of the RNA polymerase so the transcription can take place.
On the other hand, the presence of glucose, diminishes the concentration of cyclic adenosine monophosphate (cAMP), so it can´t join the enzyme catabolite activator protein, so both of them won´t join the DNA, and won´t increase the production of ?-galactosidase, which allows the cell to hydrolyse lactose and release galactose and glucose. In this case, glucose doesn´t have an effect because there is no lactose, so the production of ?-galactosidase won´t even take place.
b. I would advice him to only use lactose, for the explanations made above.
c. Bicoid, because it was the only one that allowed the production of GFP.
d.Bicoid, because it was the only one that allowed the production of GFP over Kruppel.
e. This proteins speciffically bind to nitrogenous bases.
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