Advance Molecular Biology (LS6421, 2003) Part I-2
I.
T. Y. Lin
Initiation of transcription
1. The groups of factors that act in conjunction with RNA polymerase II
(1). General factors
(2). Upstream factors
(3). Inducible factors
2. The nucleosome
(1). Unwrapping of nucleosomal DNA
a. First phase (~40 bp) - reversible
b. The second phase- irreversible melting
c. Cooperative binding of factors to nucleosomal target sites
(2). Histone octamer movement
a. The histone octamer (intact) transferred from DNA in front of the
polymerase to DNA behind it.
b. Histone octamer transfer differs from disassembly of the histone
octamer mediated by histone-binding proteins, nucleoplasmin.
3. Eukaryotic RNA polymerase
Ion-exchange column (Roeder and Rutter)
Two inhibitory compounds, one is a-amanitin, the toxin the
poisonous mushrooms Amanita phalloides (the death cap) and
Amanita bisporigera (the death angel). Polymerase II is very
sensitive to the toxin, being reduced to half its maximal activity with
about 0.02 g/ml of -amanitin. Polymerase III is next, with a
half-maximal sensitivity of about 20 g/ml. Polymerase I is
completely resistant to -amanitin.
1
The other compound is the product of a bacterium of the
Streptomyces genus called actinomycin D. Actinomycin D inserts
itself (intercalating) into DNA regions that are GC rich and inhibits
transcription from this region of the DNA. It turns out that the
genetic information for ribosomal RNA (rRNA) is GC rich
(1). RNA polymerase I has a bipartite promoter (G·C rich, ~85%
identical)
a.
The core promoter (-45 to +20) and the upstream control element
(UCE, -180 to -107) UBF1 is specific to core promoter and UCE.
b. Once UBF1 has bound, factor SL1 binds cooperatively.
c.
SL1 consists of 4 proteins including TBP. A common feature in
initiation is a reliance on a ‘positioning’ factor that is specific for
each type of promoter.
(2). RNA polymerase III uses downstream and upstream promoters.
a.
Promoters for 5S and tRNA genes are internal (+55 - +80 for 5S).
(a). Initiation occurs at a fixed distance (~55 bp) upstream of the
promoter.
(b). The downstream boundary of the promoter is at about position +80.
b. Three accessory factors for RNA polymerase III
(a). TFIIIA is a zinc finger protein.
(b). TFIIIB that consists of TBP and two other proteins is the true
initiation factor required by RNA polIII (positioning).
(c). TFIIIC (>5 subunits >500 kD) enables TFIIIB to bind to a sequence
surrounding the transcription start point (tsp).
2
c.
Promoters for small nuclear RNA (snRNA) genes lie upstream of tsp.
(a). The proximal sequence element (PSE), OCT and the TATA element
(b). The function of TBP and its associated protein is to position RNA
polymerase III correctly to the start point.
(3). RNA polymerase II
Genes for Subunits of Yeast RNA
Polymerase II
Protein Mass
Phenotype of
Gene
(kDa)
Deletion Mutant
RPB1
190
nonviable
RPB2
140
nonviable
RPB3
35
nonviable
RPB4
25
conditional
RPB5
25
nonviable
RPB6
18
nonviable
RPB7
19
nonviable
RPB8
17
nonviable
RPB9
14
conditional
RPB10
8.3
nonviable
RPB11
14
nonviable
RPB12
7.7
nonviable
(4). Core Subunits:
In the three core subunits, RPB1, RPB2, and RPB3, the first two are
homologous to the E. coli ' and  subunits, respectively. Regions
of the protein sequence of RPB1 and RPB2 are homologous to these
two prokaryotic proteins.
RPB3 has homologies to the prokaryotic  subunit.
The core proteins are found in the holoenzyme in the ratio of 1:1:2
(RPB1:RPB2:RPB3), reflecting relatedness to their prokaryotic
counterparts.
RPB1 is the subunit on which -amanitin acts, although the exact
mechanism of inhibition is not known.
3
Common subunits: RPB5, 6, 8, 10 and 12 are the common subunits.
Nonessential subunits: two of the subunits appear to be essential only
under certain conditions: RPB4 and RPB9. Deletion mutants of
these two function well at normal temperatures, but fail to grow at
either higher or lower temperatures. RPB4 seems to be required to
keep RPB7 associated with the polymerase.
Deletion mutants of RPB7 and RPB11 are nonviable, so they are
essential. There is some evidence that RPB7 has a -like role,
being responsible for correct initiation of transcription.
4. Basal apparatus for initiation of transcription of RNA polymerase II
(1). The transcription startpoint: Py2CAPy5, the initiator
(2). The TATA box (~ -25) tends to be surrounded by GC rich sequences.
(3). TFIID (~800 kD): TBP (~30 kD) and 11 TAFs (30-250 kD)
(4). RNA polymerases are positioned at all promoters by a factor that
contains TBP that makes sequence-specific contact with DNA.
(5). The inner phase of TBP binds to DNA in the minor groove. The
outer surface is available to extend contacts to other proteins.
(7). A bend at the TATA box widens the narrow groove where TBP binds.
4
(8). Within TFIID, TAFII250 binds to TBP.
(9). When TFIIA joins the complex; TFIID becomes able to protect a
region extending farther upstream. TFIIA may activate TBP.
(10). TFIIB binds adjacent to TBP and may provide the surface that is
recognized by RNA polymerase II.
Factor
(Abbreviation)
Composition
TFIIA (IIA)
2 or 3 subunits
TFIIB (IIB)
single subunit
TFIID (IID)
TFIIF (IIF)
TFIIE (IIE)
TFIIH (IIH)
Function
stabilizes binding
between TFIID and
promoters
interaction between
TFIID and polII-TFIIF
TBP (TATA box-binding binding to TATA box
protein)
interaction with
8 - 10 TAFII's
promoter elements
(TBP-associated
and with gene-specific
proteins)
transcription factors
somewhat like sigma in
prokaryotes, this
protein causes RNA pol
2 subunits
II to bind to the
complex assembly at
the promoter
required for binding
2 subunits
and stimulation of
transcription
kinase activity
(associated kinase
complex
activates polII by
phosphorylation),
helicase activity
Step 1: Formation of the DAB complex
Factors IID binds to DNA at the promoter. The TBP part of IID can
bind by itself to promoters that contain a TATA box. In TATA-less
promoters, that have an initiator sequence, the binding requires the
TAFII's. For binding to promoters containing a GC box, this
requires TBP, TAFII's, and a factor called SP1 that binds to the GC
box.
5
TBP is shown as the saddle like structure, consistent with the X-ray
crystal reconstruction of this protein complexed to DNA.
6
(11). TFIIF consists of two subunits
a.
RAP74 has an ATP-dependent helicase activity.
b. RAP38 is homologous to bacterial sigma factor.
c.
The complex of TBP and TAFs may interact with the CTD tail of
RNA polymerase II.
Step 2: Binding of the RNA polII-IIF complex
The factor IIF associates with RNA polII in a manner similar to
sigma factor in a prokaryote. This complex now joins the
IID-IIA-IIB-promoter complex.
(12). TFIIH, TFIIJ and TFIIE are required for promoter clearance.
a.
TFIIH
(a). Activities of an ATPase, a helicase
(b). A kinase activity that can phosphorylate CTD tail of RNA
polymerase II.
(c). Phosphorylation of the CTD of RNA polymerase by TFIIH is needed
to release RNA polymerase II.
(d). TFIIH is involved in DNA repair and may play a role in elongation.
b.
TFIIE and TFIIH are required to allow polymerase movement.
Step 3: Binding of IIE and IIH
The final step in what is called the preinitiation complex is the
joining of IIE and IIH. IIE appears to stabilize the complex, while
IIH has two activities: kinase (addition of phosphate groups) and
helicase (DNA unwinding).
7
RNA polymers II binds as an unphosphorylated form (called II-A)
but the form that carries out transcription is phosphorylated in its
C-terminal domain (II-O). This phosphorylation (at serines and
threonines) is catalyzed by a kinase activity associated with IIH.
(13). The CTD may coordinate processing of RNA with transcription.
a. The capping enzyme binds to the phosphorylated CTD.
b. Some splicing factors bind to CTD.
c. Some components of the cleavage/polyadenylation apparatus.
Step 4: Elongation
A factor called TFIIS is seen to stimulate the elongation reaction for RNA
polymerase II. It may be that factor IIF also has a role in elongation.
5. A connection between transcription and repair
(1). The template strand of DNA in a transcribed gene is preferentially
repaired when DNA is damaged.
a.
Mfd recognized a stalled RNA polymerase and directs DNA repair to
the damaged template strand (the excision-repair mechanism).
(a). Mfd displaces the ternary complex of RNA polymerase from DNA.
(b). Mfd causes the UvrABC enzyme to bind to the damaged DNA.
b. The ERCC3 and TFIIH
(a). TFIIH may associate with a kinase at initiation. The kinase catalytic
subunit belongs to a group of kinases involved in cell cycle control.
(b). Another complex associated with a large group of proteins coded by repair
genes (a subunit recognizes damaged DNA and an endonuclease).
(c). The large subunit of RNA polymerase is degraded when the enzyme
stalled at sites of UV damage.
8
6. Promoters for RNA polymerase
(1). Short sequences at -30, -75, -90 -basal element.
(2). TATA box is a crucial positioning component of the core promoter.
(3). The CAAT box (-75) determines the efficiency of the promoter
a. The CAAT box can interact with CTF and the factors CP1 and CP2
(gene specific).
b. The CAAT box can interact with C/ERP prefers GCAAT, ACF
prefers CCAAT.
c. The Oct-2 is a tissue-specific activator, while the Oct-1 is ubiquitous.
d. Oct-1 is the only octamer-binding factor in nonlymphoid cells.
e. Oct-2 binds to octamer to activate the immunoglobulin  light gene.
f. CAAT-displacement protein (CDP) prevents other factors from
binding to CAAT-box.
Upstream Elements:
The GC box (GGGCGG) and the CAT box (CCAAT) are upstream
elements.
Initiator Sequence:
Not all eukaryotic genes have initiator sequence elements at their
start point. Initiatos appear to have the consensus sequence
PyPyAN(TorA)PyPy. Such sequences can function as very simple
promoters, although at a low rate of transcription.
Downstream Elements:
These elements are only known by the fact that experimental
alteration of them changes the transcription of the genes.
The promoters tell the polymerase where to start. However, there
are other sequences that affect the rate of transcription.
Enhancers and Silencers:
In eukaryotic cells there are sequences that influence the rate of
transcription by interaction with specific proteins. Sequences that
increase the rate of transcription are called enhancers.
9
The two 72 bp regions are the enhancer elements. Notice the GC
boxes and the TATA box. In this case, the enhancer elements are
located near the transcription unit.
Silencers decrease the rate of transcription or, in some cases, prevent
a region from being transcribed at all.
(4). The GC box (-90, GGGCGG) is recognized by SP1.
(5). The global repressor Dr1/DRAP1 binds to TBP.
(6). CAAT is a target for regulation in sea urchin.
a.
Two CAAT are found in the promoter of histone H2B gene that is
expressed only during spermatogenesis.
b. Only CAAT-binding factors from testis can bind to the CAAT box.
c.
In the embryonic tissues, the CAAT-displacement protein (CDP)
binds to the CAAT boxes to prevent the TF from recognizing them.
7. Enhancers contain bi-directional elements that assist initiation.
(1). Upstream activator sequences (UAS) in yeast.
(2). An enhancer in the virus SV40.
a.
A region contains two identical sequences of 72 bp each; repeated in
tandem ~200 bp upstream of the start point of a transcription unit.
b.
The 72 bp repeats lie in a region of hypersensitive to nuclease.
c.
Enhancers often show redundancy in function.
(4). How can an enhancer stimulate initiation at a promoter that can be
located at apparently any distance away on either side of it?
a.
An enhancer could change the overall structure of the template, e.g.
by influencing the density of supercoiling.
b. The enhancer may be upstream or downstream of the promoter.
c.
Responsibility for tissue-specific transcription may lie with a
promoter or an enhancer.
d. It could be responsible for locating the template at a particular place
within a cell, e.g. attaching it to the nuclear matrix.
e.
An enhancer could provide an entry site, a point at which RNA pol or
10
some other essential proteins associates with chromatin.
8. Transcription factors
(1). DNA-binding and activation functions in a TF may comprise
independent domains of the protein.
(2). Yeast TF GAL4: each domain of the protein folds independently into
an active structure that is not influenced by the rest of the protein.
(3). The tat-tar interaction stimulates HIV transcription: the
RNA-binding protein provides tethering function.
(4). The DNA-binding domain brings the activating domain into the
vicinity of the start point.
9. Interaction of upstream factors with the basal apparatus
(1). An upstream factor uses its activation domain to contact the basal
apparatus.
(2). Some upstream factors bind coactivators that contact the basal
apparatus.
(3). Acidic activators (negative charge) of GCN4 enhance the ability of
TFIIB to join the basal initiation complex.
11