1.                  Describe the role of the following in the initiation of transcription.  (3 pts each).

A.                 TBP

Part of TFIID, this is the TATA binding protein that interacts directly with the TATA box

 

B.                 TFIIH

Transcription factor that uses its helicase activity to melt the double-stranded DNA at the TATA box region to give access to the template for RNA polymerase II. Also has the protein kinase activity that phosphorylates the CTD of RNA polymerase II to allow it to clear the promoter.

C.                 Mediator

Large multi-subunit protein complex that is required in-vivo to initiate transcription by mediating the interaction between the specific transcription factors on upstream promoter elements or on enhancers with the general transcription factors at the TAT box.

D.                 histone acetylase

Enzymatic activity that activates transcription by attaching acetyl groups to the lysines at the amino termini of the histones, thus neutralizing the basic (+) charges, preventing them from strongly interacting with the negatively charged phosphate groups. This loosens the chromatin structure.

E.                  chromatin remodeling factor

Proteins that work to loosen the interaction of the histones with DNA transiently allowing the nucleosomes to phase differently, opening up regions of accessibility to the transcription factors.

F.                  upstream promoter elements

Located 50-500 bps upstream of the TATA Box, these are sequences that bind to specific transcription factors, regulating initiation of transcription.

G.                 micro RNAs

Synthesized as a stem-loop structure, these small RNAs are cleaved to double-stranded intermediates and then interact with RITS or RICS complexes where they become single stranded and target specific regions of either DNA (via RITS) that draw histone methylase enzymes to repress the DNA targets and form heterochromatin or (via RICS) mRNA, targeting it for degredation.

2.                  What molecular event must occur in order for RNA polymerase to continue to elongate an initiated transcript? (3 pts)

Once the RNA polymerase II has cleared the promoter and begun transcription it stalls until elongation factors and other proteins interact to hyperphosphorylate the CTD. This allows transcription to continue.

3.                  If gene X was transcribed in cell type A but not cell type B, in which cell type would it be more sensitive to DNAse digestion? Explain why. (6 pts)

Gene X would be more sensitive to degredation in cell type A because the chromatin is less densely packed around expressed genes than those same genes in cells were they are not expressed. The DNAse can then get easier access to the DNA in the less densely packed chromatin, degrading it more easily.

4.                  Protein Y is involved in regulating the concentration of molecule Z in the cell by determining the mRNA stability of molecule Z. Predict what will happen if the cell is quickly using up molecule Z. Be sure to explain the mechanism thoroughly. (6 pts)

The mRNA of Z needs to be stabilized to allow more translation. This is accomplished by having molecule Y bind to the stem-loop sequences in the 3’ UTR that signal nucleases for degredation of the mRNA. By binding to these signals, the nucleases are not recruited and the mRNA not degraded. More Z can then be made.

5.                  Describe the chemical modification at the 5’ end of eukaryotic mRNAs . (6 pts)

This is called the Cap. It is a guanine-containing nucleotide that is methylated at the N7 position and attached to the first nucleotide of the mRNA by a 5’ to 5’ triphosphate linkage. In vertebrates, the 2’OH of the ribose in both the first and second nucleotides of the mRNA is also methylated.

6.                  How is poly-A tail addition coupled to termination of transcription in eukaryotic transcriptional processing? (8 pts)

The RNA polymerase II transcribes an AAUAA sequence that signals for polyadenylation about 30 nucleotides from the signal, usually following a CA. The RNA pol II continues past this region and also transcribes a GU-rich region of RNA transcript and probably continues even further. Various factors bind to the AAUAA and the GU-rich region and cause endonucleases to cleave at the poly-A addition site, usually following a CA. Then polyA polymerase binds and makes a short poly A tail of about 20 nucleotides. Poly A binding proteins bind to that and stimulate addition of another 20 poly As more quickly. More poly A binding proteins bind and stimulate again, until the poly A tail is from 200-2000 A residues long.

7.                  Two transesterification reactions occur during the splicing process in eukaryotic cells. Describe these chemical reactions. Do not include any discussion of the snurps in this answer. (8 pts)

First the branch-point A’s 2’OH attacks the first intron-exon junction, breaking the phosphodiester bond and covalently attaching to the 5’G of the first intron nucleotide in a 5’-2’ phosphodiester bond. Then the 3’OH now on the end of the exon thus released attacks the 5’ phosphate of the next exon, causing the intron-exon junction to break and uniting the two exons in a 3’-5’ phosphodiester bond.

8.                  Describe the role of the following in facilitating the correct splicing of eukaryotic mRNAs. (3 pts each)

A.                 UI

Binds to the consensus sequence surrounding the 100% conserved GA at the 5’ end of the intron through complementary base pairing mediated with the U1 small nuclear RNA. It can then interact with U2 to bend the intron in such a way as to facilitate the first transesterification reaction.

B.                 U2

Binds through the small nuclear U2 RNA component to the consensus sequence surrounding the branch-point A but does not bind to the A itself. This causes the A to bulge out, facilitating its attack in the first transesterification reaction.

C.                 U2AF

Binds to the pyrimidine-rich region in the intron which lies between the branchpoint A and the 5’ end of the intron. It facilitates the binding of the U2 snurp to its binding site as described in B.

D.                 SR proteins

Bind to the exonic splicing enhancers within the exons on either side of the intron to be removed. This marks the exons and helps  U1 and U2AF to find their respective binding sites within the intron.

9.                  Two forms of double-sex proteins are made in developing fruit flies and are responsible for the sexual phenotype of the flies. This system involves control at the level of transcription initiation and at the level of alternative splicing. Describe the entire sequence of events that occurs in developing fruit-flies and results in the formation of either the male or female phenotype. (10 pts)

First, the early sxl promoter is used in females to initiate transcription of the early sxl gene which is then normally spliced and the early sxl protein made. Males have this early promoter repressed and therefore do not transcribe the gene and make no protein.

Later, the late sxl promoter is used in both males and females and transcription of the gene progresses in both. However, the early sxl protein in females binds to the U2AF binding site in one of the introns, preventing U2AF from binding. This keeps U2 from binding and the intron is not spliced out. Instead, the next 3’ splice site is used and all intervening sequence is removed as one large intron. This removes an exon with a premature stop codon and allows the resulting mRNA to be translated into functional late sxl protein. In males, there is not alternative splicing of the late sxl transcript. Normal splicing keeps the exon with the stop codon in the mRNA. Therefore, when translated, the protein is quickly truncated an not functional.

The functional late sxl protein in females causes an analogous alternative splicing event in the transformer mRNA to occur, once again removing an exon with a premature stop codon, allowing functional tra protein to be made. Males cannot alternatively splice since they do not have functional late sxl protein and therefore do not remove the exon once again causing premature truncation of the tra protein making it non-functional.

The tra forms a complex with tra2 and Rb1 that operates like an SR protein on the dblsx mRNA. It binds to an exon and activates an otherwise weak 3’ splice site, helping U2AF to bind there. Males do not activate this weak site because they do not have tra. Therefore, the females remove the intron at the weak splice site, keeping the next exon, while the males look for a normal 3’ splice site further down and thus remove a larger intron that contains the exon retained in the female. Both mRNAs are functional and when translated make dblsx proteins that differ in sequence between the female and male forms. The female form represses transcription of male-specific genes and the male represses transcription of female-specific genes. This causes either a female or a male phenotype in the flies.

10.              Illustrate the secondary structure of a transfer RNA molecule. Indicate on your diagram the following: amino acid binding site, anticodon, polarity, all stem-loop structures. (12 pts)

See page 310, figure 8-1.

11.              Describe what is meant by the term wobble as it relates to the translation process. (5 pts)

Wobble is the term for stable association between nucleotides at position 1 of the anticodon of a tRNA and position 3 of a codon in mRNA that is not according to the normal Watson-Crick base-pairing rules.

Explain what influence this wobble phenomenon had on the evolution of the genetic code. (3 pts)

Since the amino acid carried by tRNAs that can wobble as described above will be used during translation, it was necessary that the genetic code not depend upon the wobble position when determining the codon for a particular amino acid. If that position is irrelevant in the codon, it will not matter if the tRNA can wobble. It will still be carrying a correct amino acid, determined by positions 1 and 2 of the codon with which it interacts during translation. This leads to degeneracy whereby there can be more than one codon for the same amino acid, but the differences lie in position 3 of the codon, which is irrelevant.