o If Ser2 phosphorylation is also lost, the polymerase pauses until re-phosphorylation by a non-TFIIH
kinase occurs
o Ser7 of the heptad repeat in the CTD is phosphorylated during transcription
▪ This raises the number of potentially regulated sites to 8, which is strong evidence for a CTD
code.
o Ser5 helps recruit the capping complex.
- There is evidence for distinct phosphorylated forms of the promoters, which may recruit different CTDKs
o Different kinases are recruited based on the combination of serine and tyrosine that are
phosphorylated
o e.g.: the loss of Ser7 from the repeats prevents 3’end processing of U2 snRNA transcripts, thus
preventing U2 expression
- CTD phosphatase:
o FCP1 was the first CTD phosphatase to be characterized.
▪ It is necessary for CTD phosphorylation in vivo.
▪ Yeast cells with temperature sensitive mutations have severe defects in poly(A) mRNA
synthesis at the nonpermissive temperature (i.e. it is essential for cell viability)
▪ FCP1 directly recognizes the CTD, but interacts with a site of RNAPII distinct from the CTD
▪ It hydrolyses phosphoserines (Ser2), and thus dephosphorylates the CTD directly
▪ The recognition of the CTD by the phosphatase is based on random access and is not driven
by POLII conformations
▪ Human FCP1 can stimulate elongation by RNAPII, and helps to recycle RNAPII at the end of
the transcription cycle by converting RNAPIIO into IIA for another round of transcription
o Other CTD phosphatases specific for Ser5:
▪ SCPs (Small CTD Phosphatases): The expression of SCP1 inhibits activated transcription from
a number of promoters, and may play a role in the transcription from initiation/capping in
processive elongation
▪ Ssu72: a component of the yeast cleaving/polyadenylation factor (CPF) complex. Plays a role
in the recycling of RNAPII and termination
- CTD phosphatase regulation:
o Pin1 is a peptidyl-prolyl isomerase that influences the phosphorylation status of the CTD by
inhibiting FCP1 and stimulating CDC 2 and cyclin B
▪ It binds to a subset of proteins and plays a role as a post phosphorylation control is
regulating protein function
▪ The up regulation of Pin1 is implicated in certain cancers
▪ The down regulation of Pin2 is implicated in Alzheimer’s disease
▪ Inhibitors of Pin1 may have therapeutic implications for certain diseases
o SSu72 can regulate sister chromatid cohesion and the separation of duplicated chromosomes during
the cell cycle
o SCPs function in silencing neuronal gene expression (capable of
silencing neural genes)
o FCP1 is disease related (cataracts and facial dysmorphisms)
Transcription Regulation
Mechanisms of Transcription Activation
- Transcription Factors are often dimers: this provides more combinations
of available TFs (can form combinations for specific binding domains) and
increases the regulation of transcription (monomers will not activate
transcription)
o The affinity of binding between a protein and DNA varies with the square of the free energy of
binding
o Free energy depends on the number of protein DNA contacts, doubling the contacts by using a
dimer quadruples the affinity
, o This is significant because most activators have to operate at very low concentrations
Transcription Activator SP1 (covered previously):
- Allows TF2D to bind to the DNA binding domain even if the TATA box is not present
- Most eukaryotic activators are composed of a DNA binding domain and a transcription activating domain
o DNA binding domains contain motifs such and bZIP, BHLH and zinc modules
o Transcription activating domains can be acidic (glutamine or proline rich)
- Eukaryotic activators stimulate the binding of TFs and RNAP to the promoters
Network of Functional Interactions at Promoters:
- Enhancers are distal regulatory elements that work in trans
o They usually boost transcription and can be found both upstream and downstream if the
transcription initiation complex
- protein-protein interactions between activators bound to enhancers, and TFs and RNAP to the promoters,
are mediated by the looping out of DNA in between
o DNA looping could bring the enhancers closer to the promoters, where they stimulate transcription
is a cooperative way
- There is a cooperation of cis DNA elements and trans TFs to recruit RNAPII to the TSS
o Most trans regulatory elements interact with the TSS with the aid of architectural DNA binding
proteins, which loop the DNA in such a way that the enhancer element docks onto the TF (bound to
the regulatory region near the TSS)
▪ Other hypotheses to explain the ability of enhancers to act at a distance involve supercoiling
to shorten the distance, the enhancer physically sliding along the DNA until it encounters the
promoter, or some form of DNA looping where the loop is enlarged until the enhancers
reach the promoter
Transcriptional Co-Activator CBP:
- cAMP participates in transcription activation in eukaryotes in a less direct
way than it does in prokaryotes, mainly through a signal transduction
pathway
- when the level of CAMP rises in a eukaryotic cell, it stimulates the activity of
protein kinase A (PHA) and causes this enzyme to move into the nucleus
- PKA then phosphorylates an activator called the cAMO response element
binding protein (CREB)
- CREB binds to the cAMP response element (CRE) and activates the associated
genes
- CREB causes transcription activation when bound toe CBP
- The CREB binding protein (CBP) binds to CREB with more affinity after CREB
has been phosphorylated by PKA
- CBP can then contact and recruit elements of the transcription apparatus
- By coupling CREB to the transcriptional activator, CBP acts as a coactivator
More On the Mediator
- The Mediator has no effect on transcription in the absence of the activator, but it greatly stimulates the
transcription in the presence of the activator (its an essential coactivator in all eukaryotes)
- The mediator can transduce signals from the activators bound to the enhancer regions to the transcription
machinery, which is assembled at promoters as the PIC
o It relays signals from TFs directly to RNAPII, facilitating TF dependent regulation of gene expression
o It is thus essential for converting biological inputs to physiological responses
kinase occurs
o Ser7 of the heptad repeat in the CTD is phosphorylated during transcription
▪ This raises the number of potentially regulated sites to 8, which is strong evidence for a CTD
code.
o Ser5 helps recruit the capping complex.
- There is evidence for distinct phosphorylated forms of the promoters, which may recruit different CTDKs
o Different kinases are recruited based on the combination of serine and tyrosine that are
phosphorylated
o e.g.: the loss of Ser7 from the repeats prevents 3’end processing of U2 snRNA transcripts, thus
preventing U2 expression
- CTD phosphatase:
o FCP1 was the first CTD phosphatase to be characterized.
▪ It is necessary for CTD phosphorylation in vivo.
▪ Yeast cells with temperature sensitive mutations have severe defects in poly(A) mRNA
synthesis at the nonpermissive temperature (i.e. it is essential for cell viability)
▪ FCP1 directly recognizes the CTD, but interacts with a site of RNAPII distinct from the CTD
▪ It hydrolyses phosphoserines (Ser2), and thus dephosphorylates the CTD directly
▪ The recognition of the CTD by the phosphatase is based on random access and is not driven
by POLII conformations
▪ Human FCP1 can stimulate elongation by RNAPII, and helps to recycle RNAPII at the end of
the transcription cycle by converting RNAPIIO into IIA for another round of transcription
o Other CTD phosphatases specific for Ser5:
▪ SCPs (Small CTD Phosphatases): The expression of SCP1 inhibits activated transcription from
a number of promoters, and may play a role in the transcription from initiation/capping in
processive elongation
▪ Ssu72: a component of the yeast cleaving/polyadenylation factor (CPF) complex. Plays a role
in the recycling of RNAPII and termination
- CTD phosphatase regulation:
o Pin1 is a peptidyl-prolyl isomerase that influences the phosphorylation status of the CTD by
inhibiting FCP1 and stimulating CDC 2 and cyclin B
▪ It binds to a subset of proteins and plays a role as a post phosphorylation control is
regulating protein function
▪ The up regulation of Pin1 is implicated in certain cancers
▪ The down regulation of Pin2 is implicated in Alzheimer’s disease
▪ Inhibitors of Pin1 may have therapeutic implications for certain diseases
o SSu72 can regulate sister chromatid cohesion and the separation of duplicated chromosomes during
the cell cycle
o SCPs function in silencing neuronal gene expression (capable of
silencing neural genes)
o FCP1 is disease related (cataracts and facial dysmorphisms)
Transcription Regulation
Mechanisms of Transcription Activation
- Transcription Factors are often dimers: this provides more combinations
of available TFs (can form combinations for specific binding domains) and
increases the regulation of transcription (monomers will not activate
transcription)
o The affinity of binding between a protein and DNA varies with the square of the free energy of
binding
o Free energy depends on the number of protein DNA contacts, doubling the contacts by using a
dimer quadruples the affinity
, o This is significant because most activators have to operate at very low concentrations
Transcription Activator SP1 (covered previously):
- Allows TF2D to bind to the DNA binding domain even if the TATA box is not present
- Most eukaryotic activators are composed of a DNA binding domain and a transcription activating domain
o DNA binding domains contain motifs such and bZIP, BHLH and zinc modules
o Transcription activating domains can be acidic (glutamine or proline rich)
- Eukaryotic activators stimulate the binding of TFs and RNAP to the promoters
Network of Functional Interactions at Promoters:
- Enhancers are distal regulatory elements that work in trans
o They usually boost transcription and can be found both upstream and downstream if the
transcription initiation complex
- protein-protein interactions between activators bound to enhancers, and TFs and RNAP to the promoters,
are mediated by the looping out of DNA in between
o DNA looping could bring the enhancers closer to the promoters, where they stimulate transcription
is a cooperative way
- There is a cooperation of cis DNA elements and trans TFs to recruit RNAPII to the TSS
o Most trans regulatory elements interact with the TSS with the aid of architectural DNA binding
proteins, which loop the DNA in such a way that the enhancer element docks onto the TF (bound to
the regulatory region near the TSS)
▪ Other hypotheses to explain the ability of enhancers to act at a distance involve supercoiling
to shorten the distance, the enhancer physically sliding along the DNA until it encounters the
promoter, or some form of DNA looping where the loop is enlarged until the enhancers
reach the promoter
Transcriptional Co-Activator CBP:
- cAMP participates in transcription activation in eukaryotes in a less direct
way than it does in prokaryotes, mainly through a signal transduction
pathway
- when the level of CAMP rises in a eukaryotic cell, it stimulates the activity of
protein kinase A (PHA) and causes this enzyme to move into the nucleus
- PKA then phosphorylates an activator called the cAMO response element
binding protein (CREB)
- CREB binds to the cAMP response element (CRE) and activates the associated
genes
- CREB causes transcription activation when bound toe CBP
- The CREB binding protein (CBP) binds to CREB with more affinity after CREB
has been phosphorylated by PKA
- CBP can then contact and recruit elements of the transcription apparatus
- By coupling CREB to the transcriptional activator, CBP acts as a coactivator
More On the Mediator
- The Mediator has no effect on transcription in the absence of the activator, but it greatly stimulates the
transcription in the presence of the activator (its an essential coactivator in all eukaryotes)
- The mediator can transduce signals from the activators bound to the enhancer regions to the transcription
machinery, which is assembled at promoters as the PIC
o It relays signals from TFs directly to RNAPII, facilitating TF dependent regulation of gene expression
o It is thus essential for converting biological inputs to physiological responses
