1. Describe the role of TFIIH in the initiation of transcription. (6 pts)

It works as a kinase to phosphorylate the CTD of RNA pol II, allowing it to clear the promoter complex.

It works as a helicase to melt the ds DNA at the promoter, providing the template for RNA pol II to begin the transcript.

2.        

A.        If a gene was transcribed in cell type A but not B, what would you expect the results would be if you performed a DNAse sensitivity assay on the DNA region containing the gene in both cell types. (5 pts)

The chromatin structure around the gene in cell type A would be be looser and therefore you would see increasing susceptibility to DNAse digestion as concentrations of DNAse increased. Even at fairly low levels of DNAse, the fragment containing the gene would disappear from a Southern blot result. The chromatin structure around the gene from cell type B would be more densely packed and less accessible to DNAse. Even at high DNAse concentrations, the fragment containing the gene would be present on a Southern blot result.

B.         Describe two types of regulatory proteins that help decondense chromatin during transcription initiation. (6 pts)

Histone acetylases attach acetyl groups to lysines in the NH2 termini of histone proteins, loosening the attraction between the protein and the negatively charged DNA. 

Chromatin remodeling complexes act to “push” the DNA out through the histone octamer, loosening the more compacted chromatin structure by phasing the nucleosomes out into a looser configuration.

3. Describe the role of the tar sequence in HIV in helping the virus complete the transcription of its genes. (5 pts)

It is transcribed early, near the 5’ end region of the transcript. It forms a stem-loop structure that binds to tat (an HIV protein) and the cellular protein cyclin T. This recruits cdk9 which hyperphosphoryltes the CTD of RNA pol II, allowing it to continue transcription. Additional cellular factors are also involved but you are not required to name these.

4. Choose ONE of the following and describe the mechanism. (7 pts)

1. cleavage and polyadenylation
2. capping

For A, see Figure 8-15. For B, see Figure 8-3.

5. What is the function of the following in the splicing reaction? (3 pts each)

1. U1: Binds to the sequence spanning the 3’ end of exon 1 and the 5’ end of the intron, in particular to the 100% conserved GU in the intron. This helps position the assembling spliceosome correctly.

2. U2: Binds to the sequence surrounding the branch-point A in the intron but not to the A itself, forcing it to bulge out. This facilitates the first transesterification reaction.

3. U2AF: This binds to the pyrimidine-rich tract near the U2 binding site. It assists U2 binding and therefore facilitates proper positioning of the spliceosome.

4. SR proteins: Bind to exonic splicing enhancer regions in the exons. They help mark the introns on either side of the exon by assisting the binding of U2AF to the intron on the 5’ side and assisting the binding of U1 to the intron on the 3’side.

6. Describe the molecular mechanism that allows the developing B cell to switch from producing a membrane-bound antibody to one that is secreted. (7 pts)

7. What is RNA editing? (5 pts)

This is the change in the nucleotide sequence of an RNA transcript. Our example was the apoB transcript. In liver cells, the transcript is not edited and gives rise to a longer form of the protein, apoB-100. In intestinal cells, an enzyme is active that deaminates a C to a U at one codon. This changes the codon into a stop codon. The protein derived from this edited transcript is shorter, apoB-48.

8. How does the cell guarantee that unfinished mRNAs do not exit the nucleus? (6 pts)

Only once the poly-A tail has been added will a phosphatase, Glc7, bind to the complex of proteins associated with the RNA transcript. It removes a phosphate group from another protein, Npl3 (an SR protein). This allows the exporter, TAP/Nxt1 to bind and allow the complex to diffuse through the nuclear pores to the cytoplasm. Then, a different kinase, Sky1 phosphorylates Np13 again, causing the dissociation of Glc7 and the exporter, allowing it to return to the nucleus with the help of an importer.

9. Below is a table representing a filter binding experiment, similar to Nirenberg’s. In this case, however, one of 3 possible codons was being tested. What do you conclude? (4 pts)

Radioactive filter?                                    Codon                         labeled amino acid

      No                                           CUU                            phenylalanine

      No                                           CGA                            proline

      Yes                                          CUU                            leucine

      No                                           CCC                            arginine

      Yes                                          CGA                            arginine

      Yes                                          CCC                            proline

      No                                           CUU                            arginine

      No                                           CGA                            leucine

CUU codes for leucine, CGA codes for arginine, and CCC codes for proline. The other codons do not code for the amino acid that was labeled.

10. Define what is meant by wobble and how it has influenced the genetic code. (7 pts)

Wobble is the stable non-Watson-Crick base pairing that can occur between anticodon position 1 and codon position 3. Since this allows tRNAs that have such anticodons to remain associated with a codon during translation, the genetic code has become degenerate regarding codons that can associate with such anticodons. The third base of the codon is irrelevant in these codons so that the amino acid being specified by the codon will be the same no matter if the third base is participating in a wobble relationship or not. This prevents incorrect amino acids that might otherwise be carried on these tRNAs from being incorporated during translation.

11. Outline the 2 step mechanism catalyzed by amino acyl tRNA synthetase. (6 pts)

The enzyme first binds to the amino acid and ATP. Then it catalyzes the hydrolysis of the bond between the alpha and beta phosphates, releasing PP and AMP. It uses the energy released to attach AMP to the COOH of the amino acid. It then flips this to a proofreading site.If incorrect, it releases the complex and tries again. If correct, the enzyme  binds to the tRNA and then hydrolyzes the bond between the AMP and the amino acid, using the energy released to attach the amino acid the 3’ end of the tRNA at either the 2’ or 3’ OH of the ribose component.

12. tRNAs bind to eEF1-alpha before they engage with the ribosome and mRNAs during translation. This provides an important safeguard to the translation process. Describe the role in translation played by this complex and explain why it helps insure that only the correct amino acid will enter the growing polypeptide. (8 pts)

The tRNA/eEF1-alpha-GTP complex randomly enters the A site of the ribosome. At first, only the anticodon side enters while the amino acid side cannot enter due to steric hindrance from the attached eEF1-alpha. If there are 3 stable hydrogen bonding interactions between the codon at the A site and the entering anticodon, the complex will remain there long enough for the GTP to hydrolyze to GDP. The eEF1-alpha GDP will no longer be able to remain attached to the tRNA and will leave, releasing the amino acid side from the steric hindrance. It will then be able to stably engage with the A site, using the energy that was released by the GTP hydrolysis. Peptidyl transferase activity would then be activated. If, however, an incorrect tRNA had entered, there would be insufficient hydrogen bonding between codon and anticodon and the complex would be knocked out of the A site before any GTP hydrolysis could occur.

 

13. Outline the steps that occur during translation initiation and that result in properly setting the reading frame for translation. (8 pts)

Refer to figure 4-24 in the text.

14.  Aconitase (IRE-binding protein) regulates the amount of iron in the cytoplasm of cells by effecting the amounts of two critical proteins. In detail, explain how. (8 pts).
    

Refer to figure 8-31 in the text.