MB 5.2.

Transcription in Eukaryotes

3 RNA polymerases responsible for copying DNA template

Pol I: Transcribes large ribosomal RNAs (nucleolus), rRNAs 12S, 18S,

5.8S

Pol II: Transcribes mRNA,and some snRNAs

(Nucleoplasm)
Pol III: Transcribes most small RNAs, tRNAs and RNPs involved in
processing primary RNA transcripts (Nucleoplasm)
cont'd
• Transcription is completed in the nucleus.
• Chromatin must be uncoiled and DNA made accessible to RNA
polymerase
-Chromatin remodeling
•Initiation involves a more complex set of interactions between
-Initiation factors, enhancers
– Elements within or downstream from gene
Eukaryotic RNA polymerase requires a large number of proteins to
assemble at the promoter to initiate transcription.

• These proteins are called general transcription factors (proteins

assembled on basal promoter elements)

•General Transcription Factors work to:

- Position the RNA polymerase correctly on the DNA promoter and initiate
transcription.
- Pull the DNA strand apart.
- Release the RNA polymerase once transcription has ended.
Transcription of protein-coding genes by RNA polymerase II

►Transcriptional Initiation in Eukaryotes

• RNA polymerase II transcribes pre-mRNAs (hnRNAs)
• In yeast has 12 subunits/polypeptides
• Like prokaryotes, eukaryotes require a promoter, two types

• Core promoter element (=TATA box and initiator ) (near the

transcription start, ~ -25 -30 bp)
Similar to E. coli –10
“Goldberg-Hogness or TATA Box” = TATAAAA (Common
consensus) )

• Proximal promoter elements (located upstream,~-50 to -200 bp)

“Cat Box” = CAAT and “GC Box” =GGGCGG

• Different combinations occur near different genes.

Eukaryotic promoter elements.

( initiator)
 The RNA Pol II is associated with six general transcription
factors

– Required for all RNAP II mediated transcription

– Bind to basal elements
– Required for polymerase binding, do not “turn gene on/off”
• Specific transcription factors
– Involved in regulating genes on/off, specific for gene or subset of
genes

• Designated as TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH, where "TF"
stands for "transcription factor" and "II" for the RNA Pol II.
TFIID : has 10 polypeptides

• TBP (TATA-box binding protein) and

• TAFs (TBP associated factors).
Role of TBP is to bind the core promoter with the assistance of TAFs-
Once binds at least 7 other general transcription factors bind to form pre-
initiation complex, which then binds RNAP II
TFIIH -catalyzes DNA melting.
•Before TFIIH can unwind DNA, the RNA Pol II and at least five general
transcription factors (TFIIA is not absolutely necessary) have to form a
pre-initiation complex (PIC).
PIC= RNA polymerase + TFs
Cont’d

The PIC assembly- Binding of TFs and RNA polymerase occurs

in a set order in protein coding genes.
 TBP first binds to the promoter and then recruits TFIIB to
join TFIID (and TFIIA if present). Before entering PIC, RNA
Pol II and TFIIF are bound together, which are recruited by
TFIIB. Finally, RNA Pol II recruits TFIIE, which further
recruits TFIIH to complete the PIC assembly.

IID+ IIB + RNA poly. II + IIF +IIE +IIH

Cont'd
 Binding of Activator Factors

Protein

• High-level transcription is induced by binding of activator factors to DNA

sequences called enhancers (distant control sequences on DNA)& stimulates
transcription
Model for Enhancer action

•Enhancer is the DNA element that,

upon binding with transcription
factors (activators), can enhance
transcription. It may be located either
upstream or downstream of the
transcriptional initiation
site. However, most of them are
l o c a t e d u p s t r e a m .
Silencer elements and repressor factors also exist

– Silencer is the DNA element that, upon binding with transcription

factors (repressors), can repress transcription.
• Silencer proteins
– bind to enhancer sequence & block gene transcription

In prokaryotes, silencers are known as operators, found in many

genes such as lac operon and trp operon.
In eukaryotes, the following genes have been demonstrated to
contain silencers
• Human β globin
•Human dopamine beta-hydroxylase (DBH) gene
•Brain-derived neurotropic factor gene
Elongation in Eukaryotic mRNA Synthesis

• After assembly PIC at the promoter -TFIIH use its helicase activity to
unwind DNA
• Then, RNA Pol II uses nucleoside triphosphates (NTPs) to synthesize an
RNA transcript.
• During RNA elongation, TFIIF remains attached to the RNA polymerase,
but all of the other transcription factors have dissociated from PIC.
• The carboxyl-terminal domain (CTD) of the largest subunit of RNA Pol II
is critical for elongation.
• In the initiation phase, CTD is unphosphorylated, but during elongation it
has to be phosphorylated. This domain contains many proline, serine
and threonine residues.
►Termination of Eukaryotic mRNA Synthesis

• Eukaryotic protein genes contain a poly-A signal located

downstream of the last exon. This signal is used to add a series of
adenylate residues during RNA processing.
• Transcription usually terminates at 0.5 - 2 kb downstream of the
poly-A signal, but the mechanism is unclear.
►Eukaryotic RNA Processing
• hnRNA is converted to mRNA
• Posttranscriptional modifications
– 7-methyl guanosine nucleotide cap attached 5’
• May be essential for transport out of the nucleus and protect
from 5’ exonuclease attack
– PolyA added to 3’ end
• About 200-250 nucleotides by polyA polymerase
• Signal is AAUAAA (actually for nuclease cleavage to produce
mature 3’ end of hnRNA
• Without polyA transcript is degraded

- RNA Splicing
Commonly involves ribozymes
-snRNAs, snRNPs
-Some RNAs are self-splicing
Self-Reading

• Intervening Sequences in Eukaryotic Genes

• Heteroduplex With “Loop Outs”
• Group of Introns
• Splicesomes and Nuclear Splicing
- Spliceosome Mechanism
• RNA Editing
Transcription by RNA Polymerase I
Transcription by RNA Polymerase III
Regulation of Transcription in Eukaryotes

• Degree of packing of DNA regulates transcription

– tightly packed = no transcription
– = genes turned off

• Methylation of DNA blocks transcription factors

– genes turned off
– attachment of methyl groups (–CH3) to cytosine
nearly permanent inactivation of genes
• ex. inactivated mammalian X chromosome
Histone acetylation
• Acetylation of histones unwinds DNA
– loosely packed = transcription
– = genes turned on
• attachment of acetyl groups (–COCH3) to histones
– conformational change in histone proteins
– transcription factors have easier access to genes
5.3. Regulation of Gene Expression

• The process by which genetic information flows from genes to proteins

is called gene expression
• Chromosomes carry hundreds or thousands of
genes- but only a fraction of theses are expressed
and are in use at any given time.
• In bacterial cells- only 25% (1000) of the total genes are expressed under
any particular set of growth condition.
•Each gene or small group of related genes is used to generate separate
RNA copy, when, and if it is needed.
• Housekeeping genes- are genes required for the fundamental operation of
the cell.
Why turn genes on & off in Eukaryotic organism?

• Specialization
– each cell of a multicellular eukaryote expresses only a small fraction
of its genes depending on their specialized role.
• Development
– different genes needed at different points in life cycle of an organism
(gene expression vary with the stage of development)
Embryonic genes are often expressed only at certain time , afterwards
need to be turned off permanently
• Responding to organism’s needs
– homeostasis
– cells of multicellular organisms must continually turn certain genes
on & off in response to signals from their external & internal
environment
5.3.1. Control of Gene Expression in Prokaryotes

• Proteins interacting with DNA turn prokaryotic genes on or off in

response to environmental changes.

• In prokaryotes, genes for related enzymes are often controlled together

by being grouped into regulatory units called operons.

• Operons are transcriptionally regulated as a single unit - one promoter

controls several proteins

• Regulatory proteins bind to control sequences in the DNA and turn

operons on or off in response to environmental changes
 Bacterial Operon consists of:

A) Structural genes:
- code for the enzymes themselves
- lie adjacent to one another
- turning on one gene turns on all the enzymes-producing genes
of an operon.
B) Promoter: Site of RNA Pol binding to the DNA prior to transcription
begin.
C) Operator: which typically resides adjacent to or overlapping with the
promoter serves as binding site for a protein, called the repressor.
- The repressor is an example of gene regulatory protein- a protein
that recognizes a specific sequence of base pairs within the DNA
and binds to that sequence with high affinity.
D) Regulatory gene encodes the repressor protein.
Types of prokaryotic operon:

1) The lac operon- the cluster of genes that regulate production of the
enzymes needed to degrade lactose in bacterial cells.
 Is an inducible operon-means the presence of a key metabolic
substance (lactose) induces transcription of the structural genes.
 Contains three tandem structural genes:
• z gene- encodes β-galactosidase.
• y gene- encodes for galactoside permease
• a gene-encodes for thiogalactoside acetyltransferase
Repressor protein bind to DNA only in the absence of
Lactose in growth medium.
Type of regulatory mechanisms are:

1) Positive control
2) Negative control
3) Catabolic Repression
4) Feed back inhibition
lac operon - the classical example

lacZ lacY lacA

The lac operon produces enzymes that break down lactose only
when lactose is present
OPERON

Regulatory Promoter Operator

gene
Lactose-utilization genes

DNA

mRNA

RNA polymerase
Protein Active cannot attach to
repressor promoter

DNA
RNA polymerase
mRNA bound to promoter

Protein

Inactive
Lactose repressor Enzymes for lactose utilization

OPERON TURNED ON (lactose inactivates repressor)

Repression
Derepressed or induced
Catabolic Repression

• CRP works on several operons that encode enzymes

used in catabolic pathways
– If glucose is present, cAMP is low, CRP is inactive, and
the synthesis of enzymes that catabolize other
compounds is slowed
– If glucose is low, cAMP is high, CRP is active, and the
synthesis of enzymes that catabolize other compounds
is increased.
2) The tryptophan (trp) operon:

• Is a repressible operon
• Repressor is active as a DNA binding protein only when in complex with
tryptophan, which is called as
a corepressor.
Tryptophan is absent from the bacterial growth medium: operator site is
open to be bind by RNA pol.- structural genes of trp operon transcribed
leading to the production of enzymes synthesizing tryptophan
Increased tryptophan concentration Formation of tryptophan-repressor
complex- Transcription blocked.
The trp operon
Feedback inhibition

 Is the inhibitory effect of the final product on the enzyme

involved in the first step of production when synthetic
pathway is made up of several steps and is controlled by
the activity of specific enzymes.
E.g.,
The end product (e.g., tryptophan) inhibits the first enzyme
(tryptophan synthetase TrpA) of the biosynthetic pathway
– Fast, immediate
5.3.2. Control of Gene Expression in Eukaryotes

Possible Levels of Control

•Transcriptional initiation
• RNA processing
• mRNA transport from
nucleus
• mRNA half-life
• Translational initiation
• Posttranslational
modifications of proteins
 For more details read books.
5.3.3. Analysis of Gene expression
• Monitoring gene expression is possible by measuring the level of
protein or RNA.
Detection of proteins
- Running cell extract on polyacrylamide
gel.
- Antibody-based assay
Estimation of transcriptional expression- by measuring the level of
mRNA directly – by Northern blotting (Hybridization) using a DNA
probe specific for the sequence of gene under investigation.
 Use of gene fusion with reporter genes.
Genes that are used in genetic analysis because their product are easy
to detect are known as reporter genes.
- used to detect the location of a protein or
- the presence of a particular segment of DNA
5.4. Post transcriptional events
• Prokaryotic transcription and translation is coupled.

• Eukaryotic mRNA is processed

-Capping with 7M-Guanosine
- Polyadenylation
- Splicing
• The spliceosome is the organelle responsible for
removing introns and splicing exons together
•Small ribonucleoprotein particles (snRNPs) within the
spliceosome recognize the intron-exon boundaries
Differences between eukaryotic and prokaryotic gene
expression
1. In eukaryotes, one mRNA = one protein.
(in bacteria, one mRNA can be polycistronic, or code for several proteins).

2. DNA in eukaryotes forms a stable, compacted complex with histones.

(in bacteria, the DNA is not in a permanently condensed state)

3. Eukaryotic DNA contains large regions of repetitive DNA.

(in bacteria, DNA rarely contains any "extra" DNA)

4. Much of eukaryotic DNA does not code for proteins (~98% is non-coding in humans)
(in bacteria, often more than 95% of the genome codes for proteins)

5. Sometimes, eukaryotes can use controlled gene rearrangement for increasing the
number of specific genes.
(in bacteria, this happens rarely)

6. Eukaryotic genes are split into exons and introns.

(in bacteria, genes are almost never split)

7. In eukaryotes, mRNA is synthesized in the nucleus and then processed and

exported to the cytoplasm.
(in bacteria, transcription and translation can take place simultaneously off the
same piece of DNA)