The trp operon: Bacterial cells can take up the amino acid tryptophan from their surroundings, or, if the external supply is insufficient, they can synthesize tryptophan from small molecules in the cell.  

When external supplies of tryptophan are plentiful, the cells suppress transcription of the trp operon, which encodes the tryptophan biosynthetic enzymes. When external supplies of tryptophan are not plentiful, the cells express the trp operon.

The trp operon repressor protein inhibits transcription of the genes in the trp operon. Upon binding tryptophan, the tryptophan repressor binds to a site in the promoter of the operon and represses transcription.

A. (2 pts) Why is tryptophan-dependent binding to the operon a useful property for the tryptophan repressor?

B. (4 pts) How would regulation of transcription of tryptophan biosynthetic enzymes be affected in cells that express a mutant form of the tryptophan repressor that (i) cannot bind to the DNA or (ii) bind to DNA even when no tryptophan is bound to it? Also address how tryptophan synthesis would be affected and how that would affect the cell. Please bullet point your response (i) and (ii) for each mutant for ease of grading.

Serotonin is synthesized from the amino acid tryptophan (normal cells can synthesize serotonin when they are given tryptophan). You have identified two populations of mutant cells that cannot synthesize serotonin. Each population has a mutation that has caused one gene to stop functioning. Population 1 cannot synthesize serotonin at all, even if you provide it additional nutrients. Population 2 can synthesize serotonin, but only if you give it a nutrient called 5-HTP.

a. Based on this information, draw a biochemical pathway starting with tryptophan and ending with serotonin. How does 5-HTP fit into this pathway? Be sure to indicate the location of any enzymes.

b. Describe the mutation you expect to see in each population based on your biochemical pathway

1. How does each of your mutations affect the amino acid sequences? Are the mutations missense mutations, silent mutations or nonsense mutations?
   1. Point mutation?
   2. Frameshift-insertion?
   3. Frameshift-deletion

The non mutated Sequence:

1. AUG GAG GUC UUU AAG AGA CAU UUA GAU GUA GCC CUU AGU GAU GUU UAG

Point Mutation:

• AUG GAC GUC UUC AAG AGC CAU UUA GAA GUA GCC CUU AGU GAU GUC UAG
• Translation: Met Asp Val Phe Lys Ser His Leu Glu Val Ala Leu Ser Asp Val Stop.

Frameshift Insertion Mutation:

• AUG UGA GGU UUU UAA GUA GAC AUU UAG AUG UAG CCA CUU AGU GAU GUU UAG
• Translated: Met Stop Gly Leu Stop Val Asp Ila Stop Met Stop Pro Leu Ser Asp Val Stop

Frameshift Deletion Mutation:

• AUG GAG UUU AAG AGA UUA GAU GUA GCC CUU AGU GAU GUU UAG
• Translated: Met Glu Phe Lys Arg Leu Asp Val Ala Leu Ser Asp Val Stop

1. What type of secondary structure would a peptide composed only of the amino acid Lysine form?

a) a random coil at all pH values

b) alpha helix at pH 7

c) alpha helix at pH well above 7

d) alpha helix at pH well below 7

2. During vigorous exercise ATP is hydrolyzed as follows,

ATP----> ADP +Pi + (H+)

what are the consequences of this for the structure-function of hemoglobin?

a) hemoglobin binds O2 stronger as a result of the changing pH

b) lower pH results in deprotonation of His146 which destabilizes the ion pair between monomers

c) lower pH results in protonation of His146 which stabilizes the ion pair between monomers

d) nothing happens to hemoglobin, only myoglobin is affected.

3. If you were to make a mutation to hemoglobin so that it could no longer form tetramers, what would you predict would occur.

a) impossible to predict

b) it would bind oxygen with lower affinity than before

c) it would transport oxygen better

d) hemoglobin would no longer be able to transport oxygen

1.   The following data were obtained from 3 separate enzyme kinetic experiments using 3 different substrates S1, S2 and S3 forming products P1, P2 and P3 respectively. The amount of enzyme in each reaction is 1 µM. Find out the rate and graph the data using a Michaelis-Menton and Lineweaver-Burk plots and determine the values for Km, Vmax, Kcat, and Kcat/Km. Which among the 3 substrate is best substrate for this enzyme and why? (4 points)

[S] (mM)   [P1] (mM) in 60 min   [P2] (mM) in 240 min   [P3] (mM) in 30 min
2.5   0.170   0.126   0.194
2   0.201   0.135   0.213
1.5   0.167   0.129   0.206
1   0.162   0.131   0.203
0.6   0.159   0.135   0.174
0.3   0.148   0.131   0.163
0.15   0.122   0.111   0.122
0.2   0.095   0.117   0.114
0.1   0.075   0.072   0.104
0.05   0.049   0.040   0.071

2.   Use the Michaelis-Menton Equation to calculate the missing values of [S] given below if Vmax = 5 mmol/min. Plot [S] versus V (NOT the reciprocals!). Draw line parallel to the x-axis at Vmax and extend your plotted line to show its approach to Vmax. (2 points)

[S] (mM)   V0 (mmol/min)
10   1.2
[S]1       1.7
[S]2       2.1
[S]3       2.2
[S]4       2.5

3.   Plot the below data and determine the type of inhibition of an enzymatic reaction by inspecting the graph (give an explanation) (2 points)

[S] (mM)   V0 (mM/min)   V0 with Inhibitor present (mM/min)
1   1.3   0.8
2   2.0   1.2
4   2.8   1.7
8   3.6   2.2
12   4.0   2.4

4.   Plot the below data with and without inhibitor (I) and determine the type of inhibition of an enzymatic reaction by inspecting the graph (2 points)

[S] µM   V0 (µmol /min); [I] = 0 nM    V0 (µmol /min); [I] = 25 nM    V0 (µmol /min); [I] = 50 nM
0.4   0.22   0.21   0.20
0.67   0.29   0.26   0.24
1.00   0.32   0.30   0.28
2.00   0.40   0.36   0.32

1. Which of the following is NOT a product of β-oxidation of a 20-carbon fatty acid?

2. Which pathway is NOT active in the liver in the well-fed state?

3. Beriberi is a condition caused by a thiamine deficiency. When analyzing blood samples of an individual with Beriberi, what would you expect to find elevated levels of?

4. Rotenone is a toxin that acts on the electron transport chain. When added to actively respiring mitochondria, the ratio of NADH to NAD⁺ increases while the ratio of FADH₂ to FAD does not change. What component of the ETC does it inhibit?

1. Membrane-associated translocators are responsible for importing polypeptides into both mitochondria and peroxisomes. The processes in the two organelles are different because _______________.

   		mitochondria have only one membrane translocator complex and peroxisomes have two
   		polypeptides are unfolded during translocation into mitochondria but not peroxisomes
   		mitochondrial polypeptides bind to cytosolic receptors or chaperones and peroxisomal polypeptides do not
   		peroxisome import requires signal sequences while mitochondrial import does not

2. Match each event in glycolysis with the correct description.

   First ATP investment step Second ATP investment step First step of cleavage stage Substrate-level phosphorylation

   A. Main point of allosteric regulation in the pathway B. Raises the free energy of glucose and traps it in the cell C. Transfers a phosphoryl group from an organic intermediate to ADP D. Produces two 3-carbon molecules that are similar but not identical

What is not true in the description of chloroplast?

Which of the characteristic iS NOT shared in common between mitochondria & chloroplastss?