PHARMACOLOGY SEMINAR 1

TRANSCRIPTION FACTORS THAT REGULATE TRANSCRIPTION IN

PROKARYOTES AND EUKARYOTES

SUBMITTED BY
R V SURIYA
BHARATH
3RD
PHARM.D
 TRANSCRIPTION 2
FACTORS:
 Proteins that control the rate of protein synthesis by switching some gene on
and off.
 IT WORKS BY.,
 They move from cytoplasam into nucleus
 Binds at promotor region either activator or repressor
 ACTIVATOR-> A help RNA polymerase to bind DNA also gene is transcribed
 REPRESOR-> presenting RNA polymerase at DNA binding site also gene is not
transcribed.
FACTORS THAT REGULATE TRANSCRIPTION: 3

 Promoters
 Enhancers
 Silencers
 Terminators
 Insulators
 Methylation
 PROMOTERS 4
A.)DIFF. STATE OF GENE EXPRESSION:
 Approximately 30,000 genes in humans,
 few express at high abundance (these are frequently tissue specific, e.g. globin
genes in red cells)
 few thousand express at low abundance (these frequently encode functions
needed in all cells, i.e. "housekeeping genes.“)

 B.) PROXIMAL ELEMENTS:

 No expression to BASAL
expression
 BASAL expression to HIGH LEVEL
expression

 Changes in Chromatin structure and roles of transcriptional factors have been

proposed in both the process.
 So the both process may have an important role in transcription.
 Basal transcription
 5
Is frequently studied by in vitro transcription, using defined templates and either
extracts from nuclei or purified components.
 Requires RNA polymerase with general transcription factors (e.g. TFIID, TFIIA,
TFIIB, TFIIE, TFIIF, and TFIIH for RNA polymerase II)
 Activated transcription
 Occurs via transcriptional activators interacting directly or indirectly with the
general transcription complex to increase the efficiency of initiation.
 The transcriptional activators may bind to specific DNA sequences in the upstream
promoter elements, or they may bind to enhancers
 The fact that the activators are bound to DNA that is close to the target (or
becomes close because of looping of the DNA) means that the local concentration
of that protein is high, and hence it can boost the local concentration of the
interacting general transcription factors.
GENE EXPRESSION 6
 C. STALLED POLYMERASE:
7
 RNA polymerase will transcribe about 20 to 40 nucleotides at the start of
some genes and then block at a pause site.

stalled polymerase enzyme

Activation of HEAT SHOCK GENE

 In the case of the heat shock genes, this requires heat shock transcription
factor (HSTF).
 SILENCER: 8
 Silencers are cis-acting regulatory sequences that reduce the
expression from a promoter in a manner - i.e. they have the opposite
effect of an enhancer.

Eg : silencers that prevent expression of a genes of the mating type in yeast

and
silencers at telomeres in yeast.

 It works by protein, such as Rap1, binding to DNA in chromatin.

->The SIR proteins assemble the chromatin into a large complex that
is not transcribed..
-> the H3 and H4 histones in the nucleosomes have hypo acetylated
N-terminal tails,
-> the DNA can be methylated,
 9

 the entire silenced complex is resistant to DNase digestion in

vitro, all of which are characteristic of condensed, closed
chromatin.
 The large multiprotein complex may be involve in positive
transcription factors and RNA polymerase.
 Thus silencing is a process of preventing gene expression by
packaging the gene into heterochromatin.

 Removal of a silencer leads to depression of the regulated

genes.
SILENCER: 10
 ENHANCERS: 11

 Enhancers are cis-acting regulatory sequences that increase level of

expression of a gene, but they operate independently of position and
orientation.
 The action of enhancers differs from promoters.
Eg :1) The SV40 enhancer :
The SV40 promoter for early gene expression contains a
TATA box and six GC boxes arranged in three sets of
repeated sequences. In addition, efficient
transcription requires an upstream enhancer consisting
of two72-base-pair (bp) repeats.
 Eg : 2) enhancers occurs at any position respect to 12
gene

 The position of enhancer can be anywhere in the gene, but the

promoter is always at the 5' end.
 known of enhancers 5' to the gene (upstream),
 adjacent to the promoter (like in SV40),
 downstream from the gene (some globin genes),
 within the gene (immunoglobulins) or far upstream
 within a locus control region (globin genes
 13
Eg: 3) Multiple binding sites for transcriptional activators

 The domain (e.g. A, C and B in the SV40 enhancer)

with at least two binding sites is called an enhansion.
Multiple enhansons make up an enhancer.
 TERMINATORS; 14

 A DNA sequence just downstream of the coding segment of a

gene which is recognized by RNA polymerase , as a signal to stop
transcription
 INSULATORS: 15
 An INSULATORS can stop the enhancers from binding to the
promoter ,if a protein called CTCF(instead of CCCTC) binds to it.

METHYLATION

 The additional of methyl group to the C nucleoides , prevents CTCF

from attaching to the insulators turning it off,allowed the enhancers
to bind the promoter.
 16

REGULATION OF
TRANSCRIPTION
 Activator proteins and other regulators 17

 1. Modular construction

 a. DNA binding domain: Sequence-specific, direct contact with DNA

 b. Multimerization domain: Allows formation of homo- or heter-

multimers

 c. Activation domain: direct or indirect interaction with targets (directly

or directly affecting the efficiency of transcription).
Eg G4L4 PATHGWAY:
18
 The first 100 amino acids comprise the DNA binding domain of
GAL4. A dimer of GAL4 protein binds to a 17 bp sequence with
dyad symmetry called UASG.
 The dimerization domain overlaps the DNA binding domain,
encompassing amino acids 65 to 98.
 the principle activation domain is an acidic region at the C
terminus.
 Negative regulation is achieved by binding at C terminus
 DNA LOOPING 19
 TFs bound at distant enhancers are able to interact with general TFs at the
promoter because the intervening DNA can form loops.
 The binding of specific transcriptional regulatory proteins to enhancers is
responsible for the control of gene expression during development and
differentiation.
 the immunoglobulin enhancer is active in lymphocytes, but not in other types of
cells.
 HELIX-TURN-HELIX: 20
 The helix-turn-helix motif was first recognized in prokaryotic DNA-binding
proteins, including the E. coli catabolite activator protein (CAP).
 In these proteins, one helix makes most of the contacts with DNA, while the
other helices lie across the complex to stabilize the interaction.
 In eukaryotic cells, helix-turn-helix proteins include the homeodomain proteins,
which play critical roles in the regulation of gene expression during embryonic
development.
 ZINC FINGER DOMAINS 21
 Zinc finger domains contain repeats of cysteine and histidine residues that
bind zinc ions and fold into looped structures (“fingers”) that bind DNA.
 These domains were mostly seen in TFIIIA but are also common among TFIIA
promoters, including Sp1.

Eg:
the steroid hormone receptors, which regulate gene transcription in response to
hormones such as estrogen and testosterone.
 Transcriptional activation domains
22
 Acidic
 Gln-rich
 Pro-rich

Targets of transcriptional
activators
Cohesion between activation domains and targets:

• Initially it was thought that acidic activators would act on basic targets.
• Gln-rich activators would form complementary H-bonded structures with the targets.

Structural work to date suggests that hydrophobic interactions interspersed with ionic bonds
(acidic activators) and H-bonds (Gln-rich activators) may drive the cohesion between the two
proteins.
 SOME FACTORS TO UNDERSTAND: 23

Name
5 System Binding site Quaternary DNA binding Activation Other
(top strand) structure domain domain comments

Sp1
SV40, cellular monomer 3 Zn fingers Cys2His2 Gln-rich phosphoprotein
housekeeping
genes

GAL4 yeast galactose CGGASGACW homodimer Zn2Cys6, acidic

regulon GTCSTCCG binuclear
cluster
 24

MyoD
determination CAGCT heterodimer heterodim basic- switch
of myogenesis G with er with ID: helix-loop- partners
E12/E47: inactive helix to
active; activate
or
inactivate
Glucoc cytoplasm: with
orticoid glucocorticoid TGGTACA "heat shock" 2 Zn fingers, binding of
recepto responsive genes AATGTTC proteins; close to Zn hormone
r T Cys2Cys2 finger ligand
nucleus: changes
homodimer conformation
, move to
nucleus and
activate
genes
REFERENCE: 25
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