Week 2

Question 1

The Mediator and its important role in vivo

1. consists of
2. mediator complex physically associates with to
3. Mediator stimulates
4. it is not required for
5. , it plays a critical role in stimulating

Answer 1

1. consists of 25-30 separate subunit proteins also highly conserved in eukaryotes
2. can see that the mediator complex physically associates with RNAPII to yield a stabilized holoenzyme
3. Mediator stimulates basal RNAPII transcription in vitro
4. it is not required for minimal transcription in vitro
5. it plays a critical role in stimulating activator-mediated transcription

Question 2

Biochemical isolation of the mediator complex

Experiment: what two fractions
Activator protein and what does it do
Transcriptional activator protein

Answer 2

Experiment
Fractionate nuclear extracts to purify two fractions containing RNA polymerase II activity
- 1) core polymerase (c-pol II), has general, basal
transcriptional activity
2) Also a holoenzyme complex with more
components (h-pol II), has much higher,
transcriptional activator-dependent activity
2. GAL4-VP16 = transcriptional activator protein. Addition of activator specifically leads to big spike
in transcription with holoenzyme

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Question 3

Genetic Identification of mediator complex 3 steps

Answer 3

• Identified mutations in RNA polymerase II in budding yeast that caused growth defects when the yeast are grown at low temperature (conditional mutants – really useful genetic trick!)
• These mutant yeast strains were then
  used to isolate suppressor mutations that rescued growth at low temperatures.
  These were called SRB genes
  (suppressor of RNA polymerase B)
• Many of these suppressor mutations
  turned out to be additional mutations in
  RNA polymerase II itself, but a number of other mutations were found in genes
  encoding protein subunits of the mediator complex

Question 4

Mediator of Transcription Complex

1. what facilitate transcription and how
2. how does the mediator complex factor in this

Answer 4

Activator proteins that are bound to enhancer elements facilitate transcription by interacting with the general transcriptional machinery
* The Mediator complex is a key regulator of this interaction: serves as a bridge between DNA binding and core
* Mediator functions at the recruitment step, stabilizing activator-dependent re-initiation intermediates so that multiple rounds of transcription occur from an initial PIC

Question 5

1. Mediator provides an opportunity for
2. Mediator subunits can have unique
3. Mutations in genes encoding these subunits can
4. Mediator can interact with what independatly
5. Given the potential for specificity in regulation, the mediator complex can be a target for

Answer 5

1. Mediator provides an opportunity for regulation of transcription in specific context
2. Mediator subunits can have unique activator specificity
3. Mutations in genes encoding these subunits can severely reduce the
   transcription of enhancer dependent transcription in vitro and in vivo
4. Mediator can interact with activators and their enhancer elements independently of its interaction with RNAPII and associated basal TFs
5. Given the potential for specificity in regulation, the mediator complex can be a target for context-specific transcription.

Question 6

RNA Polymerase II elongation

1. when does RNAPII shift into an elongation phase
2. This shift correlates with the shedding of which are replaced with x called
3. other factors recruited are primarily involved in
4. Most execute their function after they have been recruited to x but they favour

Answer 6

1. Once RNAPII has initiated transcription, it shifts into an elongation phase
2. This shift correlates with the shedding of most of the general transcription factors and Mediator and these are replaced by a new set of factors, such as TFIIS and Spt5, among others - called elongation factors, which regulate elongation
3. Other factors that are recruited are primarily involved in RNA processing
   (splicing and polyadenylation)
4. Most execute their function after they have been recruited to the CTD of the large subunit of RNAPII, but they favour a phosphorylated form of CTD (unlike the factors associated with the PIC)

Question 7

1. The three main RNA processing steps depend on the CTD
2. what is lost during elongation
3. what phosphorylates S2 during elongation
4. *S2 phosphorylation increases affinity towards

Answer 7

1. (i) 5’-capping
   (ii) splicing
   (iii) polyadenylation/cleavage
   CTD stimulates each of these events independently
2. • Phosphorylation of S5 by TFIIH (Cdk7-cycH), which is associated with recruitment of capping factors, is lost during elongation
3. • P-TEFb (complex of Cdk9-cyclinT) phosphorylates S2 during elongation
4. S2 phosphorylation increases affinity towards splicing and polyadenylation factors, which are often processing the mRNA during the transcription elongation phase

Question 8

Chromatin Immunoprecipitation approach

Answer 8

1. protein complexes are cross-linked to DNA
   - formaldehyde is used to see protein-protein or protien-DNA interaction
2. The cross-linked chromatin is fragmented into ~150-300 nucleotide fragments using sonication
3. Specific fragments are purified by antibodies that recognize specific proteins (immunoprecipitation)
4. cross-linking is reversed, and purified DNA is recovered
5. recovered DNA is measured and quantified
6. Amplify DNA by PCR using specific primers, run reaction products on gel for quantification, or use quantitative PCR approaches

Question 9

Role of beads in CHip

Answer 9

In immunoprecipitation, antibodies are coupled to beads, and these beads are incubated with the sample mixture of interest
* The antibodies selectively bind your protein of interest, and tether that protein and cross-linked DNA
(in the case of ChIP) to the beads
* The beads and everything attached with them can then be separated from the rest of the solution
(either using a magnet if the beads are magnetic, or by centrifugation and pelleting the beads)
* Whatever is not attached to the beads is still in solution and can be removed, and the beads can also be washed several times to further remove remaining impurities
* After several washes, purified material is removed from the beads by disrupting antibody-protein
interaction (this is called elution)

Question 10

3 possible chip controls

Answer 10

1. fragmented input genomic DNA that is generated prior to immunoprecipitation
2. Purified DNA from a non-specific control antibody, antibody that binds another protein, or pre-immune serum during the creation of the antibody (non-specific antibody control)
3. Purified DNA that was incubated with beads only (no-antibody or beads only control)

Question 10

Formation of 5’ cap

1. The first processing step of the precursor mRNA transcript occurs after
2. A three-step enzymatic reaction does what
3. what does not have cap structres
4. three important functions of the cap

Answer 10

1. The first processing step of the precursor mRNA transcript (also called the pre-mRNA) occurs after ~20-30 nucleotides have been synthesized by RNAPII
2. A three-step enzymatic reaction adds a cap structure to the 5ʹ end of all mRNAs consisting of a modified guanine structure: called 7-methyl G - 5ʹppp5ʹ - N (m7G)
3. RNA transcribed from RNAPI or RNAPIII do not have cap structures
4. The cap serves three important functions:
   ® through its interaction with the cap-binding complex (CBC) it ensures proper
   export to the cytosol from the nucleus
   ® prevents 5ʹ-3ʹ RNA exonuclease digestion
   ® serves as a docking site for translational machinery/ribosome

Question 10

Role of TFIIS in elongation

1. • Stimulates the
2. rate of RNAPII along the sequence
3. TFIIS also helps RNAPII in proofreading as well as reactivating x by (2 ways)
   4.

Answer 10

1. Stimulates the overall rate of elongation by limiting the length of time that RNAPII pauses (or is arrested) during transcription
2. RNAPII does not transcribe through all sequences at the same rate and so it can encounter sequences that require more time to transcribe, and therefore it stalls periodically
3. TFIIS also helps RNAPII in proofreading the transcript as well as reactivate the stalled
   enzyme:
   a) by aiding in the reverse reaction of NTP incorporation (i.e. phosphodiester bond cleavage) at the active site of the RNAPII enzyme ( allows Pol II to backtrack)
   b) by stimulating an inherent RNase activity of RNAPII (not part of the active site of transcription) allowing an alternative approach to removing misincorporated NTPs
   through local (and very limited) RNA degradation (chew back to transcribe again)

Question 10

1. model to study transcription
   elongation in a physiologically relevant context
2. Under normal conditions, pol II is believed to be bound
3. Heat shock then causes

Answer 10

1. Drosophila heat shock genes are a beautiful model to study transcription
   elongation in a physiologically relevant context
2. Under normal conditions, pol II is believed to be bound to the transcription start region
3. Heat shock then causes poised RNAPII at hsp70 locus to escape the
   promoter resulting in productive elongation

Question 11

Role of P-TEFb in transcription elongation

1. • P-TEFb (CDK9-cyclinT) is the target of. and what does DRB do
2. P-TEFb phosphorylates (two things) and what does this do
3. • P-TEFb thus overcomes what to do what and why is this important

Answer 11

• P-TEFb (CDK9-cyclinT) is the target of DRB !
  *DRB inhibits ATP-dependent kinase function
  *P-TEFb phosphorylates serine 2 of the RNA pol II CTD repeats
  *remember from last module that this is an important step in transition of pol II
  elongation
  *P-TEFb phosphorylates the Spt5 subunit of DSIF
  *This modification changes DSIF from a negative regulator of elongation
  to have a positive effect
• P-TEFb phosphorylates the RD subunit of NELF
  *This causes NELF to leave polymerase
  *phosphorylation of RNAPII by P-TEFb alleviates inhibition of NELF
• P-TEFb thus overcomes the influences of the negative factors and allows
  RNAPII to enter productive elongation
  Reversing the early block in RNAPII elongation is another mechanism for regulating rate
  of transcription (as opposed to PIC assembly being rate limiting)

Question 11

Importance of elongation - Transcription of HIV genome

1. • Efficient transcription of HIV genes requires
2. x element on the HIV transcript contains sequences recognized by y
3. y positions and activates what and this allows for what
4. Therefore, TAR+Tat independently recruit what and can overcome

Answer 11

1. Efficient transcription of HIV genes requires viral protein encoded by the tat locus encoding a sequence-specific RNA binding protein (Trans-activator of Transcription)
2. the 5ʹ TAR (Trans-activating Response) element on the HIV transcript contains sequences recognized by viral Tat protein and cellular Cyclin T protein
3. Cyclin T positions and activates CDK9 to the CTD of RNAPII, allowing for efficient and productive transcription elongation
4. Therefore, TAR+Tat independently recruits P-TEFb, and can overcome
   premature termination of transcription by NELF and unphosphorylated Spt4/5
   (together referred to as N-TEF)

Question 11

3 ways to regulate elongation

Answer 11

1. promoter clearance
2. 5’ capping and pausing
3. productive elongation