1. What are the major differences between prokaryotic and eukaryotic transcription?
A. Eukaryotic transcription is carried out by 3 RNAP compared to 1 in Prokaryoutes.
B. RNAPs of eukaryotes cannot initiate transcription without the help of general
transcription factors (needed for binding to DNA).
C. The primary transcript in Eukaryotes contains introns which have to be spliced to
produce the mature mRNA. The process of maturation also includes adding a 5’ Cap
and a 3’ poly A tail.
D. The chromatin structure in eukaryotes limits access of the enzymes to the genes.
This helps regulate the process of transcription. (0.01% transcriped at any given
moment, approximately 3 genes).
N.B: RNAP IV plays a role in gene silencing. In plants, it transcripes specific RNAs that are
involved in gene silencing.
2. Which hypothesis regarding eukaryotic RNAPs was proven with α-amanitin and
actinomycin D (be specific)?
Hypothesis: There are 3 RNAPs in Eukaryotes (I, II and III). It also proved they played
different roles in transcription.
ά-amanitin is an inhibitor of RNAP II. It’s a lipid soluble molecule (can cross the membrane)
found in mushrooms (Death cap and Destroying angel). It can be used to identify the type
of RNAP in action since RNAP I is insensitive to it, RNP II is highly sensitive and RNAP III is
only slightly sensitive.
Actinomycin D is an antibiotic produced by Streptomyces that intercalates into GC rich
regions of DNA inhibiting their transcription. (RNAP can no longer bind to them) RNAP I is
the most sensitive to it. It can be used in chemotherapy.
3. Which genes are transcribed by RNAP I? RNAP II? RNAP III? (details required
for proceeding lectures)
a. RNAP I transcripes large rRNA precursors such as 28S, 18S, and 5.8S
b. RNAP II transcripes hnRNA which undergoes processing to produce mRNA (inhibition of
RNAP II by ά-amanitin is fatal).
c. RNAP III transcripes 5S rRNA and tRNA mainly.
Hint: RNAPI = rRNA. RNAPII = mRNA RNAPIII = tRNA.
4. What does CTD stand for? Explain the role of CTD tail in eukaryotic gene
CTD stands for Carboxy-Terminal Domain.
The large subunit of RNAP II (only) contains the CTD which can be in a:
a. Un-Phosphorylated (II a): involved in binding the RNAP to the promoter.
b. Phosphorylated form (II o): involved in elongation as well as splicing, addition of the 5’
CAP and the 3’ Poly-A tail (i.e. all post-transcriptional modifications) and termination. 5. How would you define enhancers? What are their characteristics? What is the
difference(s) between enhancer and upstream control element?
a. An enhancer is a sequence of DNA (cis element) that binds to transcription factors like
activators or silencers to influence the rate of transcription. They can have multiple binding
sites for different transcription factors and are independent of the orientation and location
(i.e. upstream, downstream, intron, exon).
b. Upstream elements are only found upstream and in proximity to the promoter.
N.B: RNAP are made up of 8-14 subunits. 5 of which are common to all three RNAPs as they
perform similar functions.
RNAPs of Eukaryotes require general TFs to initiate transcription.
CTD tail: Stretch of 7AA that are repeated multiple times (>10 but number is specific to the
organism). 5 of the 7AA have an OH group and therefore, the tail can be phosphorylated
(into OP) and is hydrophilic. Refer to Q4 for functions.
Eukaryotes have NO operons.
Instantaneous mRNA level is controlled by transcription rate as well as degradation rates.
6. Explain the use of reporter genes for estimation of promoter strength.
Reporter genes are genes whose products are easily assayed. They are incorporated after
the promoter whose activity is to be measured. When the promoter is activated and gene
transcription starts, the reporter gene is also transcriped and the level of formation of its
product is proportional to the strength of the promoter.
7. Explain briefly 5’ deletion series. What kind of information do they reveal?
5’ deletion is done by mutating or deleting parts of the promoter starting from the 5’ end.
The level of transcription of the reporter gene is compared to that of the control (original
promoter with no deletions). By comparing the two levels, the function of the area deleted
can be deduced (if the transcript level decreases, the area deleted is important for one or
more steps of the transcription process, however, if the level increases, the area deleted is
an inhibitory region). It may be used to locate promoter, enhancer, repressor and silencer.
E.g.: Lac Z gene produces β-galactosidase which hydrolyses X-Gal (yellow) into a colorless
galactose and blue 4-chloro-3-bromo-indigo. (yellow>>blue)
8. Explain the modular nature of RNAP II promoters.
RNAPII promoters are composed of core promoter and enhancer/silencer (cis elements).
There are variety of different core promoters and elements and none is vital for all
promoters. Depending on the situation and desired outcome, a combination of a promoter
and cis element(s) are brought together. Cis elements include TATA box, initiator, and CG
N.B: Promoter is a sequence that must be located in the vicinity of the start point and are
required for initiation of transcription. Most class II promoters contain a core element as
well as a regulatory element. Core promoter: the minimal set of elements required for accurate in vitro transcription
initiation by RNAP II. They are necessary for recruitment, binding and proper positioning of
1. TATA box (position at ~ -30): It binds TBP which is involved in initiation of
transcription for RNAP I and III as well as TATA less promoters.
2. Initiator (Inr; on the transcription start site).
3. Downstream element (DPE; downstream).
4. TF II B recognition element (BRE)
Regulatory elements influence transcription rate through binding of regulatory proteins.
They influence the positioning of the RNAP. However, they are sometimes necessary for
initiation of transcription. They are classified in respect to the distance from a core promoter
and activity (+ vs. – effect on transcription) of the element
Upstream elements which are close to the promoter (GC box, CCAAT box and promoter-
Enhancers and silencers (everywhere, orientation is irrelevant)
Also boundary elements and insulators
For details, refer to Lecture 21 notes.
9. Draw a diagram of RNAP II promoter (show all types of elements that we have
mentioned in class).
Enchancers: closely arranged elements that bind transcription factors, can be everywhere,
orientation independent. Upstream elements: dispersed element, close to promoter,
Core promoter element
(e.g. TATA box)
10.Explain the tissue (cell type) specificity of eukaryotic cis elements. DNA content of every cell is the same, therefore, cis elements as well as the promoters are
the same. However, Transcription factors are specific to every cell and control the function
of the cis elements and therefore, the transcription of genes. Therefore, they are
responsible for the specificity of the cells.
11.Knowing that different genes may have the same promoter and enhancer
elements and that different transcription factors contain the same structural
features, how would you explain transcriptional specificity?
Although different TF contain the same structural features, the way they are used and what
they bind to (modular nature of promoters) allow for transcriptional specificity. It is the
combinatorial control using TF that controls how TF brings out the effect. Also a response
has its corresponding cis element and similar responses have similar cis elements.
E.g. the heat shock proteins are transcriped in response to high temperature as well as
heavy metals. Heat shock response elements (HRE) and metal responsive elements (MRE)
are the TFs that control the transcription of the gene.
N.B: genes that respond to multiple conditions have multiple cis elements.
12.If you know the binding site for certain transcription factor (TF), outline
experiments you would use to purify this TF and to assay its activity.
The gene would be shredded into pieces and would go through beads containing TF