N.B: Transcription initiation is controlled by 4 molecular mechanisms:
a. Direct influence of TFs on GTFs.
b. Chromatin remodelling caused by TFs.
c. Regulation of concentration and activity of TFs by hormones and other signals.
d. Protection of active gene promoters from methylation.
1. Describe the role of histone acetylation/deacetylation in regulation of
Histones have N terminal tails of 20-40 AA which contain positively charged lysine
groups. When Lysine is acetylated (by acetyle transferrase aka histone acetylases
HATs), the positive charge is neutralized. Since DNA is negatively charged and the
neutralization effect of the positive lysine is now lost, repulsion occurs leading to the
formation of a less condensed chromatin (Euchromatin). Binding sites are now
exposed and transcription is activated. (GTFs bind followed by RNAP)
Deacetylation on the other hand, increases the positive charge on the lysine residues
leading to lesser repulsion between the DNA strands and therefore, a more
condensed heterochromatin structure. Binding sites are now inaccessible for TFs and
transcription is inhibited.
N.B: Deacetylases are present in co-repressor complexes and coactivators have HAT
2. Describe the role of chromatin remodelling complexes in regulation of
Histone acetylation isn’t sufficient for activation; nucleosomes are still intact and
have to be repositioned to expose the promoter elements. This requires ATP and a
remodelling complex such as the SWI/SNF complex of yeast and the RSC complex.
SWI/SNF proteins alter the structure of the nucleosomes core. The SWI proteins also
move the nucleosomes on DNA resulting in exposing the enhancers. I.e. the SWI/SNF
complex helps chromatin remodelling and removal of histone octamers from
NOT IN NOTES: RSC is a 15-subunit complex with the capacity to remodel the
structure of chromatin. It exhibits a DNA-dependent ATPase activity stimulated by
both free and nucleosomal DNA and a capacity to change nucleosome structures. It
is at least 10-fold more abundant than the SWI/SNF complex and is essential for
mitotic growth. (wiki)
3. Describe an influence of activators and repressors on assembly of
initiation complexes. **
Activators/repressors do not bind to promoters but enhancers/silencers. Initiation
complexes (ex. TFIID and such) bind to these sequences rather than the promoter to
stimulate/inhibit transcription. Competition, inhibition, direct repression.
a. Repressors can inhibit gene activation by binding to a site that overlaps an
activator binding site (repressor incapable of DNA binding replaces activator →
inactive heterodimer) b. Repressor protein binds at site adjacent to activator site and interferes with AD of
c. Repressor binds upstream, interacts through mediator with GTFs to inhibit
d. Corepressors recruited that alter nucleosomes in such a way (such as
Deacetylation) as to inhibit transcription.
4. Explain the role of enchancesomes and architectural proteins in regulation
of transcription initiation?
Enhanceosomes are protein complexes that bind to the enhancers. They help initiate
specific gene expression by binding tissue specific TFs, non-specific TFs and co-
activators along with the GTFs. (combinatorial control) Enhanceosomes work
together with TFs forming a DNA loop that helps improve the interaction between
initiation complex at enhancer and RNAP at promoter. The looping works better if the
distance between enhancer and core promoter isn’t too short.
Architectural proteins bind to specific binding sites leading to a change in the shape
of a DNA in a control region so other proteins can interact with each other and with
GTFs to stimulate transcription.
5. Explain the role of mediators and insulators in regulation of transcription
Mediators and insulators are types of TF. Mediators work with TF of enhancer and
core promoter to form the initiator complex and to help RNAP be positioned
correctly. Insulators define the effective range of transcription so that effects of
silencers or enhancers apply only to the target section of DNA.
Details: Mediator complexes help bridge different protein complexes necessary for
the assembly of the pre-initiation complex (RNAP + GTFs) in some genes. Also, by
allowing the binding of different activators, mediators control the rate of re-entry of
RNAP II. (Slow re-entry or fast re-entry)
If the enhancer is >50Kb away from the promoter, insulators are required to help
control their activity. Insulators setup boundaries between DNA domains preventing
activation/repression of genes that are close by but unrelated to that specific
activator/repressor. They also prevent gene silencing by controlling the boundaries
between Heterochromatin and Euchromatin.
6. What is the role of DNA methylation in regulation of transcription initiation?
Both DNA and histones can be methylated leading to formation of inactive chromatin.
Methylation leads to change in conformation which prevents binding of TFs to
enhancer or promoter. Therefore, to initiate transcription, the promoter has to be
un-methylated. However, an un-methylated promoter isn’t necessarily active. (other
factors required) Demethylation causes cytosine to become thymine through DNA
7. Describe how steroid hormones regulate transcription (one example is
enough; remember: nuclear receptor could be in cytoplasm or in nucleus,
already bound to DNA). ** Transcription of TF is under external signal regulation and such signal includes
steroid. Receptors for steroid on nuclear receptors bind to the ligand and increase
abundance of TF. Steroid control TF and TF control transcription.
Details: Steroid hormones bind to steroid hormone receptors (aka nuclear receptors
= TFs) forming a complex. The complex then binds to another copy of itself forming
a homo-dimer. The dimer then binds (may be transported from the cytoplasm to the
nucleus) to its response element on DNA which is part of the promoter. Binding of
the complex activates or represses transcription.
8. What is (are) the role(s) of transcription factors during development?
Different TFs are synthesized at different stages of development. These TFs help
control transcription by forming mediators and transcription complexes that bind
different GTFs and lead to a more stage specific gene transcription. Therefore, TFs
help control the specificity of gene expression. (Cis elements are the same at all
9. Describe the role of TBP during transcription (think about promoters for all
three eukaryotic RNAPs and TATA-less RNAPII promoters – how do
RNAPs bind to them; also, think about coordination of activities of all three
TBP (TATA Binding Protein) is a positioning factor for all RNAP that helps initiate
transcription. No matter which RNAP is transcriping, TBP allows the polymerase to
bind to its promoter through:
a. SL1 complex for RNAP I
b. TFIID for RNAP II
c. TFIIIB for RNAP III
d. TATA-less promoters through use of TAFII150 and 250 for elements
and through Sp1 for GC boxes.
When these form the initiator complex, RNAP is recruited so that it may bind to the
promoter correctly. TBP help control how strongly RNAP binds to the promoter and
the rate of transcription. #1 commitment factor (once TBP binds, trans
TBP also helps coordinate activities of all 3 polymerases by binding to other
10.What is the role of Sp1 protein? What is the role of SL1 protein? What do
they have in common?**
SP1 is a TF involved in gene expression in TATA-less promoters. They help bind
TFIID to a promoter containing GC box.
SL1 is a complex assembled by UBF – TBP + 3 RNAPI specific TAFs (TBP-associated
factors). It is mainly responsible for initiating transcription and trans complex
formation Both proteins help position RNAP at the start site of transcription, they
both have TBP and TAF parts in common and both bind to upstream elements. Also,
they both allow assembly of TAF.
N.B: Each RNAP has different promoters, requires polymerase specific GTFs and
recognizes different DNA control elements. RNAP I & III do not require ATP hydrolysis to initiate transcription
RNAPI RNAPII RNAPIII
Single type of promoter: Discussed in 20-23. 3 types of promoters: 2
core promoter + upstream Include TATA box, Initiator internal (1 for tRNA and 1
control element (Inr), downstream for 5S rRNA) and an
elements (DPE) and TF II upstream promoter for
B recognition element snRNAs.
(BRE) as well as
11.What is unusual about type 1 and 2 promoters for RNAPIII polymerase? **
Type 1 and 2 are internal promoters that exist near structural genes. Their effect is
minimal, the upstream elements are the ones whose change that control the
efficiency of transcription. They also form part of the coding sequence.
12.Describe the mechanism of attenuation of the Trp operon. Explain the
importance of this mechanism for a bacterium?
Attenuation is premature termination of transcription. A Leader sequence is the
sequence between the promoter-operator region and the CDS. It consists of a leader
RNA (regions 1 & 2) and an attenuator (point of choice between elongation and
termination- regions 3&4).
Trp leader sequence contains 2 Trp residues. In excess Trp, nascent mRNA is quickly
formed. Regions 1 and 2 may form a loop (without polyU) pausing transcription
transiently only. Ribosome initiates translation at the start codon near the 5’end.
The Trp leader peptide (contains 2 Trp residues) is translated easily due to the
abundant Trp available. Finally, the loop involving regions 1 and 2 melts, a new loop
involving the attenuator sequence (region 3 and 4) is formed. This loop includes a
PolyU region and RNAP terminates transcription before reaching the Trp structural
genes (Rho independent termination).
This mechanism is important to save energy. If Trp is present in excess, formation of
structural Trp genes will be a waste of resources and vice versa.
N.B: the mechanism above only occurs in prokaryotes as it involved transcription
and translation occurring at the same site which is only valid for prokaryotes.
13.Could you imagine a mechanism similar to the mechanism of attenuation
of the Trp operon in Eukaryotes? Explain your reasoning.
No, because transcription and translation are conducted at different parts in
eukaryotes, mRNA must be modified (ex. spliced) before it can be translated
and the slow mechanism (relative) of eukaryotes would not allow this process
to be effective.
14.Describe two distinctly different ways in which the trp operon is controlled
by the overall availability of tryptophan. **
1. The mechanism described in 12. Also, in low Trp conditions, nascent mRNA is
quickly formed. Ribosome initiates translation but stalls at Trp codons in region 1 until some Trp charged tRNA is formed. Region 1 can’t bind to region
2. However, region 2 forms a loop with region 3 (without polyU). RNAP
continues transcription and passes the attenuator. Structural genes of Trp
operon are expressed.
2. The aporepressor-corepressor mechanism: An aporepressor for Trp is
transcriped by a separate promoter from that of the Trp structural gene.
However, the aporepressor can’t bind to the operator of the Trp gene due to
differences in conformation. In excess of Trp, Trp acts as a corepressor, binds
to the aporepressor causing a change in its conformation and forming the
repressor. The repressor has the correct conformation and can bind to the
operator and inhibit further transcription.
15.Describe the mechanism responsible for shutdown of the trp operon
when a plentiful supply of free tryptophan is available.
Same as 14. Attenuation and repressor.
16.Describe the mechanism by which the leader-attenuator region fine tunes
the extent of transcription of the structural genes in the trp operon when
Trp is available (but not to the point to activate the repressor).
When tryptophan is available, tRNA becomes more available as well. This influences
the rate of leader sequence, which controls the loop formation. Thus, when there is
tryptophan, the leader behaves in a way that creates region 3-4 loop to cause
17.Describe rho-dependant transcription termination
Rho is a hexameric RNA helicase. It has an RNA binding domain (N-term) and
ATPase (C-term) activity. It binds to rut site (rho utilization site) on mRNA and
tracks along the RNA until it catches up with the RNAP (requires hairpin or stem loop
to stop RNAP until rho catches up). Rho unwinds DNA-RNA hybrid using ATP. mRNA,
Rho and RNAP are all released.
N.B: Rho has no affinity for RNAP
18.Describe rho-independent transcription termination
They occur at self complementary GC rich sites (dyad symmetry). Transcription of
these sites lead to the formation of a stem loop structure followed by series of U
residues. The loop interacts with RNAP causing it to pause. The AU bonds at the 3’
end of the new RNA are very weak and melt during the RNAP pause on the loop. RNA
chain is released and DNA strands re-anneal. RNAP dissociates from the DNA.
1. Describe the process of mRNA cleavage and poly-adenylation. (Don’t
forget the role of CTD tail) – not on Version 2- Q19B replaces it.
mRNA Cleavage: Group I and II are self-splicing introns. i.e. RNA functions as a ribozyme and splices
itself. Some group II organellar introns code for proteins maturases which increases
the rate of self-splicing by stabilizing 3D lariat conformation.
Group III can’t splice thems