1.       All of the DNA repair mechanisms we discussed end the same way but begin differently. What are the types of lesions recognized during base-excision repair, nucleotide excision repair, and mismatch repair?  (6 pts)

Base-excision repair corrects mismatched base pairs due to deamination or depurination events. Nucleotide excision repair corrects bulky adducts such as thymine dimers. Mismatch repair corrects incorrect base pairs that are the result of errors during DNA replication and the repair occurs very soon after replication.

2.       The importance of chromatin structure in controlling initiation of transcription was demonstrated using the enzyme DNAse.  Describe what was done in this study, illustrate what the results looked like, and explain them. (10 pts)

Two cell lines, 14-day erythroblasts that express the globin gene and MSB cells that do not were subjected to increasing amounts of DNAase. The DNA from both cells was then cut by restriction enzyme Bam H1 and the resulting DNA fragments were analyzed by agarose gel electrophoresis and Southern Blot analysis. If the DNA containing the globing gene were not degraded, the Bam H1 would produce a fragment of 4.6kb in size that hybridized during the blotting procedure to the globin probe. When the results were analyzed, the DNA from the 14-day erythroblasts showed less and less of the fragment with increasing amounts of DNAase in the procedure indicating degredation. That from the MSB cells showed no degredation even at the highest concentration of DNAase used. This correlated to the expression level of the gene in the cell types. In the cell that expressed the gene, the gene sequences were more susceptible to degredation suggesting a looser chromatin structure. In the cell that did not express the gene, no degredation suggested its chromatin structure was denser and inhibited access to the DNAase.

3.       Answer the following about transcription initiation. (4 pts each)

A.      Where is the TATA box located and why is it important?

Approximately 25-30 nucleotides of the +1 site for transcription. It binds the general transcription factors that allow RNA polymerase II to bind and initiate transcription.

B.      To what regulatory sequences do the specific transcription factors bind and where are these sequences located?

Bind to the upstream promoter sequences located 50-200 or so nucleotides upstream from the +1 site and also bind to enhancer sequences located far from the start site, often 1-kb away on either side.

C.      What two important activities are associated with TFIIH?

Helicase  activity that unwinds the double-stranded DNA at the promoter to allow RNA polymerase II to begin transcription. Also,  protein kinase activity that phosphorylates the CTD of RNA polymerase II to allow it to clear the promoter.

4.       How does chromatin become decondensed when a gene is being activated for transcription? (8 pts)

A chromatin remodeling complex phases out nucleosomes from the 30 nm fiber by pushing the DNA out of the nucleosome and a histone acetylase attaches acetyl groups to the amino terminal lysines of the histones, loosening the attachment of the histones to the DNA. See figure 7-43 for a specific example.

5.       What do the following factors, enzymes, or sequences do during processing of the RNA transcript? (3 pts each)

A.      CFI and II

Cleaves the phosphodiester bond that creates the 3’ end of the transcript and site of polyadenylation.

B.      PAP

Binds and stimulates the cleavage (see above) and attaches the poly-A tail to the 3’ end

C.      Guanylyl   transferase

Hydrolyzes GTP to GMP and PP and catalyzes the attachment of GMP to the diphosphate end of the RNA transcript in a 5’-5’ linkage.

D.      2’O methyl transferase

Catalyzes the attachment of a metyl group from S-adenosyl methionine to the 2’O of the ribose in the first and sometimes second nucleotides of the transcript.

E.       AAUAAA

Poly-A signal that serves as the binding site for CPSF and begins the assembly of the cleavage and polyadenylation factors

6.       Outline the CHEMICAL reactions that remove an intron during splicing. (8 pts)

See Figure 8-8.

7.       Describe how the SR proteins direct the splicing machinery to the introns, protecting the exons from accidental removal. ( 8 pts)

SR proteins bind to the ESE regions in an exon. They interact with one another and with two other splicing related factors and facility their interaction with the introns. Specifically, U2AF is assisted in binding to the pyrimidine-rich region of the intron to the 5’ side of the exon. U2AF then can assist the binding of U2 to the branchpoint-A region (not the A itself) within that intron. The SR proteins also help U1 to bind to the intron to the 3’ side of the exon. Specifically, the U1 will bind to the consensus sequence spanning the exon/intron junction including the 100% conserved GU.  This is illustrated in figure 8-13.

8.       How do the U1 and U2 snurps facilitate the accuracy of splicing. (8 pts)

U1 snurp interacts through its RNA component with complementary sequences that span the exon/intron junction at the 5’ side of the intron, including the 100% conserved GU. U2 interacts through its RNA component with complementary sequences that surround the branchpoint A within the intron but not with the A itself, causing the A to bulge out and facilitating the first transesterification reaction. Both snurs also help the remaining snurps to assemble into the spliceosome.

9.       Match the following to the correct statement below. Can be used more than once. More than one answer can apply to a statement (include all that do). (2 pts each)

Early sxl                                               tra

Female dsx                                         Male dsx

Tra2                                                       Late sxl

Early sxl promoter                           Late sxl promoter

Rbp1

A.      Members of an ESE binding complex_tra, tra2, Rbp1________________________________

B.      Not activated in male flies _early sex lethal promoter_____________________________

C.      An RNA-binding protein _sxl early, sxl late, Rbp1, tra2___________________________

D.      Represses female-specific genes __male dsx_____________________________

E.       Represses a splicing event _early sxl, late sxl____________________________________                                                

10.   Choose one to answer: (8 pts)

A.      Describe how the cell insures that only fully-processed mRNAs exit the nucleus.

B.      Describe how a cell switches from making a membrane-bound antibody molecule to one which is secreted.

A.      See figure 8-22

B.      See figure 24-18

11.   This question will be given to you in class. (7 pts)

Wobble is the non-Watson-Crick base pairing that is allowed between the number one position nucleotide of the anticodon on tRNA and the number 3 position nucleotide of the codon on mRNA. This base-pairing would allow a stabe enough interaction between codon and anticodon during translation to insure that the amino acid carried on the tRNA would be used during the translation process and incorporated into the polypeptide. Therefore, for those codons that would be interacting with a tRNA that caused a wobble interaction, the third nucleotide of the codon evolved to be irrelevant and essentially, only positions one and two of these codons specify the amino acid.