General transcription factors and activators 1

MLAB 230 - Molecular Biology

Topic 3: general specific

General transcription factors and activators

present in every gene and determines how
required for the polymerase does this
to bind the promoter polymerase bind
WHAT IS and how much
NEEDED FOR does it induce
TRANSCRIPTION transcription

ENHANCE
TRANSCRIPTION
If activators are missing, we have minimal, basal levels
of transcription
 General transcription factors and activators 2

General transcription factors

activator binds enhancer and
stabilizes general factors complex
enhancer Gene-specific
transcription
factor = activator
promoter à Higher levels of
transcription
TATA

No general transcription factors

à  Polymerase not recruited
à  No transcription

More activators
are involved
TFII
à +++ Higher
D levels of
transcription

General transcription factors

à  They recruit the polymerase
à  Basal levels of transcription
If a repressor binds on the silencer, it will prevent the binding of everything
else --> no transcription
 General transcription factors and activators 3

General transcription factors

Ø  The general transcription factors combine with RNA polymerase to form
preinitiation complex.

Ø  This complex is competent to initiate transcription as soon as nucleotides are

available.
melt the DNA
Ø  This tight binding involves the formation of an open complex in which the
DNA at the transcription start site has melted to allow polymerase to read it.

Ø  There are three polymerases (class I, II, and III) => three initiation
complexes.

Ø  We will start with class II.

 General transcription factors and activators 4

The class II pre-initiation complex

Ø  The class II complex contains:
•  Polymerase II
•  TFIIA
•  TFIIB
•  TFIID -- TATA box binding protein :: 1st to bind

•  TFIIE
•  TFIIF
•  TFIIH

Ø  Many studies show that the complex interact in a specific order.

cannot bind simultaneously
 General transcription factors and activators 5

The class II pre-initiation complex

Ø  TFIID with help from TFIIA binds the TATA box, forming the DA complex
Ø  TFIIB binds next, causing minimal perturbation of the protein-DNA interaction
 General transcription factors and activators 6

The class II pre-initiation complex

TATA box
Ø  TFIIF helps RNA polymerase bind to a region extending from -34 to +17.

join the complex DAB as a complex Pol/F

Ø  The remaining factors bind in this order: TFIIE and then TFIIH, forming the
DABPolFEH preinitiation complex.
Ø  In vitro, the participation of TFIIA is optional.
 General transcription factors and activators 7

The class II pre-initiation complex

Ø  Experimental inc conc of
Pol2
identification of the
pre-initiation complex

lighter

Ø  (Gel analysis on next

slide)

individual
proteins
 General transcription factors and activators 8

The class II pre-initiation complex – Gel analysis

Ø  Gel mobility shift assay using TFIIA, TFIID, TFIIB, TFIIF, and RNA Pol II.

Ø  When TFIID and A added to the promoter, a DA complex formed (lane 1).

Ø  When TFIIB is added to D and A, a new, DAB complex formed (lane 2).

Ø  In lane 3, labeled D+A+B+F, all four factors were present, but no RNA
polymerase => No detectable difference with the DAB complex.
Ø  => TFIIF does not seem to bind independently to DAB.

Ø  When increasing amounts of polymerase were added (lanes 4–7), two new
complexes appeared, including polymerase and TFIIF (including or not
TFIIA).
Ø  Then quantity of TFIIF decreased (lanes 8–12) => decrease in DABPolF.

Ø  => RNA polymerase and TFIIF are needed together to join the complex.

Ø  Without TFIID (lane 13), no complex => TFIID is the first factor to bind.

Ø  Lane 14, shows that TFIIB was needed to add polymerase and TFIIF.

Ø  Lane 15, demonstrates that leaving out TFIIA made little difference.
 General transcription factors and activators 9

The class II pre-initiation complex

Ø  Starting with the DBPolF complex
and then adding TFIIE and TFIIH in
turn => larger complex, with reduced
mobility, with each added factor.

Ø  The final pre-initiation complex

formed in this experiment was
DBPolFEH.

Ø  Last four lanes => leaving out any of

the early factors (polymerase II,
TFIIF, TFIIB, or TFIID) prevents
formation of the full pre-initiation
complex.

Ø  => The order of formation of the pre-

initiation complex is as follows:
TFIID (or TFIIA & TFIID), TFIIB,
TFIIF & polymerase II, TFIIE, TFIIH.
 General transcription factors and activators 10

Structure and function of TFIID

Ø  TFIID is a complex protein containing a TATA box-binding protein (TBP) and
13 core TBP-associated factors (TAFs).

When a protein is highly conserved --> very critical for function

changing or mutating it will impair function

Ø  TBP is highly conserved. The conserved domains encompass mainly the C-

terminal 180 amino acids mostly basic. since DNA is acidic
Ø  A truncated form of TBP containing only these 180 amino acids is enough to
bind the TATA box region of a promoter.
Ø  TBP in TFIID binds to the minor groove of the TATA box.
Ø  TBP is versatile since it also acts with TATA-less polymerase II promoters,
TATA-less polymerase III promoters, and TATA-less polymerase I promoters
 General transcription factors and activators 11

Structure and function of TFIID

Ø  Most of the TFIID TAFs are highly conserved in eukaryotes
Ø  Two main functions:
•  Interacting with gene-specific transcription factors A, B, Pol2...
•  Interacting with core element promoter TATA, GC box..

Ø  Different combinations of TAFs are required to respond to various

transcription activators. variable TAFs and combinations based on promoter type

Ø  Some exceptions:
•  Some studies suggest that TAFs do not appear to be universally required for
transcription of class II genes.
•  Some promoters in higher eukaryotes respond to alternative proteins (TBP-
related factor [TRF1] or TBP-like factor [TLF]) or can be stimulated with a TBP-
free TFIID complex.
 General transcription factors and activators 12

Structure and function of TFIID

Ø  In a TATA-containing promoter:

•  TBP can bind by itself to the TATA box of

this promoter (top)

or

•  It can also bind in the company of all the

TAFs in TFIID (middle)

or

•  And it can bind with a subset of TAFs

(bottom).
 General transcription factors and activators 13

Structure and function of TFIID

Ø  In a TATA-less promoter containing an
initiator and a downstream promoter element:

•  TBP cannot bind by itself to this promoter

(top)

•  The whole TFIID is competent to bind to the

TATA-less promoter through interactions
between TAF1 and TAF2 (middle)

•  TAF1 and TAF2 are sufficient to tether TBP to

the initiator and DPE (bottom).
 General transcription factors and activators 14

GC box and CAT box are part of the

Structure and function of TFIID upstream elements

Ø  In a TATA-less promoter containing GC

boxes:

•  TBP cannot bind by itself to this promoter

(top)

•  The whole TFIID can bind to this promoter

through interactions with Sp1 bound at the
GC boxes (middle)

•  TAF1, TAF2 and TAF4 are sufficient to anchor

TBP to Sp1 bound to the GC boxes (bottom).

Sp1 are activators that bind to GC boxes

specific for some genes
 General transcription factors and activators 15

Structure and function of TFIIF

Ø  TFIIF plays one major role: it helps binding the polymerase to the DAB
complex.

Ø  TFIIF is composed of 2 subunits, RAP 70 and RAP 30 (RNA polymerase II

Associated Protein).

Ø  It has two essential functions:

•  It helps the polymerase going into the growing complex

•  It can reduce non-specific interactions between the polymerase and DNA

 General transcription factors and activators 16

Structure and function of TFIIE and TFIIH

Ø  TFIIE binds the complex after polymerase+TFIIF.
Ø  TFIIE is a tetramer composed of 34 and 56 KDa proteins, both required for
binding and transcription stimulation.

Ø  TFIIH is the last factor to join the complex.

Ø  It has 9 subunits and plays 2 major roles: Pol is activated by phosphorylation
•  Phosphorylation of the CTD of the RAN
NA polymerase CTD: C-terminal domain in subunit 1

•  DNA helicase activity that is essential for transcription. It will cause full melting
of the DNA at the promoter and facilitate promoter clearance.
make the promoter clear again

Ø  TFIIE and TFIIH are both not essential for formation of the open promoter
complex, or for elongation, but they are required for promoter clearance.
important for allowing polymerase to move
--> promoter clearance
 General transcription factors and activators 17

The general transcription factors

Ø  Role in initiation, promoter clearance, and elongation.

excess phosphorylation -->

stabilizes complex

1) DNA unwinding
2) CTD phosphorylation
promoter clearance
 General transcription factors and activators 18

Regulation of polymerase II elongation

Ø  RNA polymerase II doesn’t transcribe at a steady rate. Instead, it pauses
sometimes for a long time before resuming transcription.

Ø  These pauses tend to occur at certain pause sites.

Ø  If the pause is too long, the polymerase cannot recover on its own, and
needs help from elongation factors.

Ø  TFIIS is an elongation factor: it stimulates elongation by limiting polymerase

pausing (but not initiation).

Ø  Such pausing can be detected during in vitro transcription by gel

electrophoresis, the results show discrete bands that are shorter than the full-
length.

Ø  TFIIS minimized the appearance of these short transcripts, indicating that it

minimized transcription arrest.
 General transcription factors and activators 19

Transcription elongation factors

Ø  At time –3 min, they added DNA and RNA polymerase.
Ø  At time 0, they started the reaction by adding NTPs. heparin impairs RNA pol functioning
Ø  At time +1 min, they added heparin to bind any free RNA polymerase (so all
new NTP incorporation is due to elongation, not new initiation).
Ø  At time +2.5 min, they added TFIIS (red) or buffer (blue) as negative control.

In elongation studies,
heparin in added to
make sure that the
incorporation of NTPs Dashed vertical lines:
are strictly due to elongation
and not to transcription fold stimulation of total
starting all over again by RNA synthesis by
free RNA pol
TFIIS.

-3
Heparin binds FREE RNA pol
If at -3min we added 200 RNA pol, then
at 0 mins 150 RNA pol started trancription
so at 1min when heparin is added, 50
RNA pol are inactivated
 General transcription factors and activators 20

How can TFIIS restart arrested transcription?

Ø  During arrest, the RNA polymerase has
backtracked so far that the 3’-end of the
nascent RNA is no longer in the enzyme’s
active site.
cannot continue
Ø  Instead, it is extruded out through a pore. since 3' is no longer
on active site
Ø  With no 3’-terminal nucleotide to add to, the
polymerase is stuck. RNase activity
within pol
Ø  So TFIIS activates the RNase activity in
RNA polymerase II, which cleaves off the generate a
extruded part of the nascent RNA and new 3' end

creates a new 3’-terminus in the enzyme’s

active site.
Ø  TFIIS also contributes to proofreading of
transcripts by stimulating the inherent
RNase in the RNA polymerase to cleave off
mis-incorporated nucleotides.
 General transcription factors and activators 21

The class I transcription factors

Ø  The pre-initiation complex that forms at rRNA promoters is much simpler than
the polymerase II complex.

Ø  Class I complex involves:

•  Polymerase I
•  SL1 = TFIID since has TBP

•  UBF (Upstream-Binding Factor)

Ø  SL1 is composed of TBP and three TAFs, and binds on core promoter
Ø  TBP is identical in SL1 and TFIID but the TAFs are completely different.

Ø  UBF binds on the upstream promoter element of class I promoters.

Ø  UBF and SL1 act synergistically to stimulate transcription.
 General transcription factors and activators 22

INTERNAL

The class III transcription factors

Ø  TFIIIA binds to the internal promoter not 5S rRNA since TFIIIA is missing

of the 5S rRNA gene and stimulates

its transcription.
Ø  Unlike TFIIIA, TFIIIB and TFIIIC act
not only in 5S rRNA gene transcription
but in all Polymerase III related genes.
Ø  TFIIIC and TFIIIA bind the internal
promoter and help TFIIIB bind to a
region just upstream of the
transcription start site.
Ø  TFIIIB then remains bound and can
sponsor the initiation of repeated
rounds of transcription.
Ø  TFIIIB contains a TBP and associated
TAFs required for the transcription of
non-classical class III genes as well. TFIID=SL1=TFIIIB
 General transcription factors and activators 23

Role of TBP in transcription factors

Ø  The assembly of the pre-initiation
complex on each kind of eukaryotic
promoter begins with the binding of
an assembly factor to the promoter.

Ø  With TATA-containing class II

promoters, this factor is TBP. Other
promoters have their own factors.

Ø  The specificity of the TBP depends

on its associated TAFs.

Ø  TRF1 substitutes for TBP at least in

some preinitiation complexes in
drosophila.
 General transcription factors and activators 24

Activators – reminder
Gene-specific
transcription
factor = activator
à Higher levels of
transcription

No general transcription factors

à  Polymerase not recruited
à  No transcription

More activators
are involved
à +++ Higher
levels of
transcription

General transcription factors

à  They recruit the polymerase
à  Basal levels of transcription
 General transcription factors and activators 25
big family: all-activators

Activators
activators repressors
Ø  Activators can either stimulate or inhibit transcription by RNA pol II.

Ø  They have a structure composed of at least two functional domains:

•  DNA binding motif

•  Transcription-activating domains bind to other proteins or factors that initiate transcription

Ø  Many have also a dimerization domain that allows activators to bind to each
other forming homodimers, heterodimers, or even multi-dimers.

Ø  Some have binding sites for effector molecules such as steroid hormones.
 General transcription factors and activators 26

Classes of activators
Classes of DNA binding motifs
Ø  Zinc-containing modules:
•  Zinc fingers
•  Zinc modules
•  Modues containing two zinc ions and six cysteines

Ø  Homeodomains (HDs)

Ø  bZIP and bHLH motifs

basic zipper and basic helix loop helix

Classes of transcription-activating domains

Ø  Acidic domains cannot be DNA binding motif since - and - repel

Ø  Glutamine-rich domains

Ø  Proline-rich domains
 General transcription factors and activators 27

Zinc fingers
Ø  Zinc fingers are composed of an antiparallel β-
strand, followed by an α-helix.
Ø  The β-strand contains two cysteines, and the α-
helix two histidines that are coordinated to a
zinc ion.
Ø  The coordination of amino acids to the zinc
helps form the finger-shaped structure.
Ø  The specific recognition between the finger and
its DNA target occurs in the major groove.
 General transcription factors and activators 28

Nuclear receptors
Ø  Nuclear receptors interact with a variety of steroid and other hormones.
Ø  They must bind to this effector (their ligand) in order to function as activators.
Ø  The glucocorticoids receptor is representative of this group.
Ø  It has a DNA-binding domain with two zinc-containing modules.
Ø  One module contains most of the DNA-binding residues. The other module
provides the surface for protein-protein interaction to form dimer.
Ø  One extra base pair between half-sites in the DNA target prevents specific
interactions with one of the glucocorticoid receptors monomers.

4 domains:
transactivation domain (green)
DNA binding domain
(orange)
effector dimerization domain (middle)

repressor
since dimer, the DNA sequence
should be repeated twice for both receptors
to bind

effector binding domain (black)

 General transcription factors and activators 29

Homeodomains
Ø  Homeodomains are DNA-binding domains
found in a large family of activators.
Ø  Their name comes from the gene regions
called homeoboxes in which they are
encoded.
Ø  Homeodomain proteins are members of
the helix-turn-helix family of DNA-binding
proteins.
Ø  Each domain contains three α-helices, the
First and the second form the helix-turn-
helix motif, the third serving as the
recognition helix. ON DNA
Ø  The N-terminus of the protein forms am
arm that inserts into the minor groove of
the DNA.
 General transcription factors and activators 30

The bZIP and bHLH domains

Ø  bZIP and bHLH domains have 2 functions: DNA binding and dimerization.
Ø  The ZIP and HLH parts of the names that refer to the leucine Zipper and
Helix-loop-helix parts of the domains, are the dimerization motifs.
Ø  The b refers to a basic region that forms the majority of DNA-binding motif.
Ø  bZIP proteins dimerize through a leucine zipper, putting adjacent basic
regions of each monomer in a position to embrace the DNA target site.
Ø  bHLH proteins dimerize through a helix-loop-helix motif which allows the
basic parts of each long helix to grasp the DNA target site.

DIMER

b ZIP

bZIP bHLH
 General transcription factors and activators 31

Function of activators
Ø  Basal levels of transcription are frequently insufficient at weak promoters, so
cells have activators to boost this basal transcription to higher levels by a
process called activator recruitment.
Ø  Activator recruitment leads to the tight binding of RNA polymerase
holoenzyme to a promoter.
Ø  Activators stimulate binding of general transcription factors and RNA
polymerase to a promoter, two hypotheses are possible:

The truth may be a

combination of the
two hypotheses
 General transcription factors and activators 32

Action at a distance
a.  An activator can bind to an enhancer and change the topology or shape of
the whole DNA molecule opening the promoter up to general factors.

b.  An activator can bind to an enhancer and then slides along the DNA until it
encounters the promoter where it can activate transcription.
 General transcription factors and activators 33

Action at a distance
c.  An activator can bind to an enhancer and by looping out DNA in between,
interacts with proteins at the promoter, stimulating transcription.

d.  An activator can bind to an enhancer and a downstream segment of DNA to

form a loop. By enlarging this loop the protein tracks toward the promoter.