FINAL EXAM M132 Spring 05
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Question 1 (10 points): 15. Describe three mechanisms by which the activity of a
transcription factor can be regulated?
Any three of the following:
Regulation of transcription of the transcription factor (TF) gene.
Regulation of the translation of the TF mRNA.
Regulation of the degradation of the TF.
Regulation of the nuclear import and export of the TF.
Regulation of the TF by small, hydrophobic molecules, such as for the nuclear
receptors.
Regulation of TF DNA binding activity by cooperative DNA-binding with a
neighboring TF.
Regulation of TF DNA-binding by interaction with another polypeptide, as for MyoD
and Id.
Regulation of the activation domain activity by post-translational modifications such
as phosphorylation.
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FINAL EXAM M132 Spring 05
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Question 2 (20 points): How does the mechanism of integration of LTR
retrotransposons differ from the mechanism of integration of non-LTR
retroransposons? What mechanism generates the short direct repeats of the
integration site sequence at each end of retrotransposons? (A diagram may be
helpful here.)
LTR retrotransposons are reverse-transcribed into double stranded DNA in the
cytoplasm. A tRNA functions as the primer for reverse transcription. The doublestranded DNA is then imported into the nucleus with the integrase protein that
functions like the transposase of DNA transposons to integrate the ends of the linear
retroviral DNA into chromosomal DNA. The reverse transcription process generates
LTRs at each end of the integrated retroviral DNA. The upstream LTR functions as a
transcription control element ("promoter" is acceptable here). The downstream LTR
functions as the polyA signal for the full-length retroviral RNA.
Non-LTR retrotransposons are bound by the ORF2 protein at their polyA-tail in the
cytoplasm and imported into the nucleus as RNA. Once in the nucleus, the ORF2
protein cleaves chromosomal DNA in a T-rich region. The T-rich DNA strand
hybridizes to the polyA tail of the retrotransposon RNA and primes synthesis of the
first strand of retroviral DNA. Chromosomal DNA serves as the primer for reverse
transcription. The promoter for non-LTR retrotranposon transcription is internal to the
non-LTR retrotransposon DNA. The weak polyA site is also internal.
For both types of retrotransposons, the duplication of sequence at the insertion site
results from a staggard cut in the double-stranded DNA at the integration site.
Synthesis of the complementary strands of the DNA at the insertion site results in
the short direct repeat of the target site sequence at each end of the inserted
retrotransposon.
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FINAL EXAM M132 Spring 05
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Retrotransposon
DS DNA
Integrase makes staggered cuts in
target DNA.
Integrase ligates DS retrotransposon
DNA to 5’ single-stranded ends of
target DNA.
retrortransposon
DNA.
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FINAL EXAM M132 Spring 05
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3. Question 3 (10 points): Alternative RNA splicing is an important aspect of
gene control in metazoans (multicellular animals). What two molecular
mechanisms can regulate the use of alternative splice sites in complex
transcription units? Give an example of each type of mechanism.
Repression of splicing (or negative regulation of splicing).
Activation of splicing (or positive regulation of splicing).
In Drosophila sex determination, the Sxl protein represses splicing of a splice
site in the Sxl pre-mRNA.
or, Sxl protein represses splicing of a splice site in Tra pre-mRNA
Tra protein activates a splice site in the Dsx pre-mRNA.
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FINAL EXAM M132 Spring 05
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Question 4 (10 points): Mutations in splice sites in human - and -globin
genes generally result in the use of alternative “cryptic” splice sites rather
than exon skipping. What mechanism can account for this? Alternatively,
a mutation within an exon of the SMN2 gene involved in the disease spinal
muscle atrophy results in the skipping of this exon in most SMN2 mRNAs.
What mechanism can account for this?
The mutation in the SMN2 exon eliminates binding by an SR-protein. This
interferes with the formation of a cross-exon recognition complex. As a result,
U2 snRNP fails to bind to the 3' splice site at the 5'-end of the exon and U1
snRNP fails to bind to the 5' splice site at the 3'-end of the exon. Since
splicesomes do not assemble at these splice sites, the exon is skipped.
In contrast, in the globin genes, mutations in splice sites do not interfere with
the binding of SR-proteins to exonic splicing enhancers in the associated
exon. The SR-proteins bound to exonic splicing enhancers stimulate the
binding of U1 or U2 snRNPs to alternative cryptic splice sites that are utilized
in place of the mutant splice site.
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FINAL EXAM M132 Spring 05
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Write legibly. No credit for wording that cannot be read easily.
Question 5 (20 points): What signals (i.e. RNA sequence) in a pre-mRNA
specify the site of polyadenylation? How is the site of polyadenylation
related to transcription termination by RNA polymerase II? What is the
mechanism that controls the change in the polyadenylation site of
immunoglobulin heavy chain µ pre-mRNA as immature B lymphocytes
develop into IgM secreting plasma cells? (A diagram of the alternative
RNA processing in the production of secreted and membrane bound 
heavy chain mRNA would be helpful.)
The site of polyadenylation is specified by a poly A site composed of an
AAUAAA upstream and a G/U-rich sequence downstream of the cleavagepolyadenylation site.
Polyadenylation does not occur at the site of transcription termination, but at a
cleavage site upstream. Cleavage and polyadenylation of the pre-mRNA
signals RNA polymerase II to terminate transcription at one of multiple
possible sites.
The secreted  heavy chain mRNA is generated by polyadenylation at a site
upstream of the poly A site for the mRNA encoding the membrane bound
form of  heavy chain mRNA. See diagram below.
The affinity of the G/U-rich sequence at the upstream secreted  heavy chain
(s) mRNA polyA-site for the CStF polyadenylation factor is very low, while
the downstream membrane bound  heavy chain (m) mRNA is a strong
polyA site with high affinity for polyadenylation factors. In immature B
lymphocytes, the concentration of CStF is sufficiently low that the s polyA
site is not used, but the strong m polyA site is used. When immature B
lymphocytes mature into plasmacells, the concentration of CStF increases to
a high enough level that the weak s polyA site is used.
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FINAL EXAM M132 Spring 05
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high CStF
low CStF
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FINAL EXAM M132 Spring 05
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Question 6 (10 points): What mechanism accounts for the sudden onset of
DNA synthesis in S. cerevisiae?
When the S-phase cyclin-CDK complexes are synthesized in late G1, they
are immediately bound by the S-phase inhibitor (Sic1). The activity of the Sphase Cyclin-CDK complexes increase precipitously when the S-phase
inhibitor is degraded suddenly. This allows the S-phase Cyclin-CDKs to
phosphorylate DNA replication initiation factors in pre-replication complexes
bound to DNA replication origins resulting in the initiation of DNA synthesis.
The S-phase inhibitor is polyubiquitinated by the SCF ubiqutin ligase after it is
phosphorylated by late G1 Cyclin-CDKs.
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FINAL EXAM M132 Spring 05
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Question 7 (10 points): What kind of mutation in a mitotic cyclin can lead to
entry into mitosis but failure to exit mitosis, i.e. failure to decondense the
chromosomes and reassemble the nuclear envelope?
A mutation in the mitotic cyclin destruction box that prevents the protein from
being recognized by and polyubiquitinated by the APC ubiquitin ligase (with
the help of the unphosphorylated Cdh1 specificity factor).
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FINAL EXAM M132 Spring 05
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Write legibly. No credit for wording that cannot be read easily.
Question 8 (10 points): Most proteins have a diameter that is much smaller
than the diameter of the central transporter of nuclear pore complexes
(NPCs). Yet proteins >60 kDa cannot diffuse through a nuclear pore. What
is thought to prevent large proteins from diffusing through the central
transporter of NPCs? (A diagram might be helpful here.)
The central transporter of the NPC is proposed to contain a molecular
meshwork of FG-nucleoporins that act like a sieve holding back proteins >60
kDa. These FG-nucleoporins are proposed to contain extended chains of
hydrophilic amino acids punctuated by hydrophobic FG-domains that
associate with each other through hydrophobic interactions, generating the
molecular meshwork. See diagram.
FG-domain
FG-Nup
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