ASSIGNMENT
Answer the following questions
You may provide your answers in a separate file or within this worksheet (save as a .pdf file) but for the latter, remember to TYPE YOUR ANSWERS IN RED (or another color of your choice)!
Part I: Gene Regulation of the trp Operon
The following questions are about the prokaryotic trp operon (outlined below), specifically addressing how the operon is regulated in response to nutritionally poor environments. Briefly, the trp operon includes a group of 5 genes (described below as genes A-E) that encode for the biosynthetic enzymes required to synthesize the amino acid, tryptophan. The structure of the trp operon is shown, including the promoter sequence which contains a regulatory sequence known as an operator. Use your knowledge of gene expression and consider the consequence for a bacterium in the following situations.
(4 points) The trp operon is expressed (i.e. turned ”on”) when cytoplasmic tryptophan levels are low, which allows the bacterium to synthesize tryptophan. However, the trp operon is repressed (i.e. turned “off”) when tryptophan levels are high. The cause of the repression is a bacterial protein known as the trp repressor.
When the trp repressor is bound to tryptophan, the protein can readily recognize and bind to the operator sequence. Why would this action prevent the expression of the trp operon?
How would a drop in the cytoplasmic levels of tryptophan allow for the expression of the trp operon? Hint: you will need to consider your answer to Q1A.
(3 points) Given what you know about gene expression, consider how much “effort” the cell puts into expressing a gene product. How would the energy requirements necessary to synthesize a protein help explain why organisms would go through the effort of regulating gene expression? In other words, why wouldn’t a bacterium express the trp operon all the time (as opposed to only expressing it when tryptophan levels are low)?
(3 points) What would happen if there was a mutation that caused the trp repressor protein to bind more tightly to tryptophan (even if tryptophan levels were low)?
(3 points) What would happen if there was a mutation in the trp repressor binding sequence (the operator sequence) that made the repressor unable to bind (even if tryptophan levels were high)?
(3 points) What would happen to the cell (when tryptophan levels are low) and there was a nonsense mutation early in the coding sequence of gene D?
(4 points) What would happen if a mutation were introduced in th domain of the Trp repressor that changed an asparagine residue to a glycine residue? Would this be more problematic for the cell when tryptophan levels were high or low?
(2 points) Do eukaryotes have operons? If not, explain how eukaryotes would regulate the expression of multiple related genes so that they are expressed at the same time.
Part II: Gene Regulation of the ara Operon
In the absence of glucose, Escherichia coli can still generate the energy necessary for proliferation by using the pentose sugar arabinose. The figure below outlines the arabinose (ara) operon, which encodes for all the proteins necessary to achieve this switch in sugar metabolism. The araA, araB and araD genes encode the enzymes for the metabolism of arabinose while the araC gene encodes a transcription regulator that binds adjacent to the promoter of the arabinose operon. To understand the regulatory properties of the AraC protein, you engineer a mutant E. coli in which the araC gene has been deleted so that you can look at how the presence or absence of the AraC protein affects the expression of the AraA enzyme.
(4 points) If the AraC protein works as a repressor, would you expect araA RNA levels to be high or low in the presence of arabinose in the araC– mutant cells? What about in the absence of arabinose? Explain your answer.Your findings from the experiment are summarized in the following table.
(4 points) Do the results indicate that the AraC protein regulates arabinose metabolism by acting as a repressor or an activator? Explain your answer
BONUS (1 point): Below is a picture of a transcription factor whose asparagine side chain (shown on the right) is contacting an adenine ring in a DNA molecule.
Which of the amino acids shown below would be able to make the same H-bonding contacts as the Asparagine? Circle your answer.
Would the substitution of the amino acid from part A change the strength of the protein-DNA complex? Why or why not
