1. Describe the general role played by the following enzymes in DNA repair. (3 pts each)

A. an endonuclease

Makes a cut on either side of the lesion on the damaged strand only.
 

B. a DNA helicase

Melts the hydrogen bonds between bases on the two strands, allowing the damaged area to be removed
 

C. DNA polymerase

Fills in the removed area (gap) with correct DNA nucleotides.
 

2. How does the RNA polymerase II enzyme clear away from the transcription initiation complex at the promoter in order to begin and continue the transcription process? (8 pts)

TFIIH uses its protein kinase activity to attach phosphate groups to the Carboxy Terminal Domain (CTD) of RNA polymerase II. This loosens the interaction between RNA polymerase and most of the general transcription factors at the TATA box, allowing it to begin moving in the 5’-3’ direction. Also, TFIIH uses its helicase activitiy to do the initial melting of the double-stranded DNA at the promoter to allow RNA polymerase to begin transcription.
 

3. Illustrate or describe in words two ways that a repressor protein might inhibit the inititation of transcription process. (8 pts)
 

Any two of the figures shown on page 406, Figure 7-49
 

4. Draw the results you would expect to see from an R-looping experiment using the following gene as the source of the DNA. (6 pts)

Answer should show double-stranded hybrid region, indicating the exons, and single-stranded looped out regions, indicating all of the introns. Also, at the 5’ end is single-stranded DNA (sequences upstream of the transcription start site) and at the 3’ end is single stranded RNA (poly-A tail).
 

5. Outline the chemical reactions that allow for the accurate removal of introns from eukaryotic transcripts. (10 pts)

See page 320, figure 6-29
 

6. What role is played by the following proteins in the creation of the 3’ end of a eukaryotic mRNA? (3 pts each)
 

A. CPSF

Binds to the AAUAAA signal on the primary transcipt. Helps position the cleavage factors

B. CstF

Binds to the GU-rich region downstream on the transcript, also helping position the cleavage factors

C. Poly-A polymerase (PAP)

Uses ATP as a source of the A nucleotides and energy needed to add the poly-A tail to the newly formed 3’ end of the transcript
 

7. Suppose that Nirenberg was doing his filter binding assay on another planet where the genetic code was read in groups of 4 nucleotides and there were only 5 possible amino acids (1-5). He was in a hurry so he decided to mix together two of the codons in the same experiment, getting the following results.

Synthetic codon ABCD mixed with synthetic codon BCDA:

Radioactive amino acid tube   Radioactive counts on filter?

 1      yes
 2      no
 3      no
 4      yes
5 no
 

What can you conclude from this experiment? (6 pts)

Amino acid 1 is coded for by either ABCD or BCDA. Amino acid 4 is coded for by either ABCD or BCDA. Amino acids 2, 3, and 5 are not coded for by ABCD or BCDA.
 
 

8. Describe the 3 important roles played by eEF2 during translation elongation. (12 pts)

A. Serves as another check on the charged tRNA, excluding a tRNA that carries an incorrect amino acid.
B. Acts as a hindrance to stable attachment of the tRNA to the A site of the ribosome, allowing time for the tRNA to be knocked out of the A site due to insufficient numbers of hydrogen bonds between the anticodon and the codon at the A site if the tRNA is not the one that should be used there.
C. Provides energy for stable attachment of the correct tRNA to the A site  by hydrolyzing its bound GTP to GDP and P, which releases energy and allows the eEF2 to leave the tRNA, allowing it to be stably attached at the A site.
 

9. Below are the stem-loop structures at the 5’ and 3’ ends of the mRNAs for ferritin and transferin receptor, under either high iron concentration in the cell or low iron concentation in the cell. Show on the diagrams where aconitase would be bound under the two conditions. (8 pts)

High iron     Low iron
Ferritin mRNA
 
 
 
 

Transferin receptor mRNA
 

Consult page 450, figure 7-105.
 
 

10. Define the following terms. (4 pts each)

A. chaperone
A protein that binds to exposed hydrophobic side chains on an improperly folded polypeptide, with the help of ATP. This allows time for the protein to fold properly. After a short time, ATP is replaced by ADP and the chaperone lets go of the polypeptide. This cycle can be repeated several times to try to have the polypeptide fold properly.
 

B. proteosome

A barrel-like structure composed of a protease-filled central core and a cap that degrades misfolded proteins and proteins marked as short-lived.
 

C. ubiquitination

Attachment of a polymer of ubiquitin proteins to a lysine side chain on a protein designated for degredation in a proteosome
 
 
 

11. Why is the genetic code degenerate? (6 pts)
 

The third base of a codon frequently can base-pair to the first base of an anticodon on a tRNA that is not the usual accepted base. This is called wobble. Because tRNAs that participate in wobble will have enough stability at the A site of the ribosome to be used in peptide bond formation, they must carry the correct amino acid specified by the codon, regardless of what base is at their wobble position. This has caused the genetic code to move to making the third base of the codon irrelevant for all codons that could have tRNAs with wobble bases with which they would interact during translation. Only bases one and two in these codons specify the amino acid.
 
 

12. Name three of the debate topics we have already heard debated in class thus far. (6 pts)
 

We have heard the first 4 debates. See the course website to read the topics. Any 3 of these can be used to answer the question.