Transcript Model for transcriptional activation

Transcription in eukaryotes
• RNA Polymerases
• Promoters
• General Transcription Factors
Model of yeast RNA polII with RNA-DNA
hybrid in the active site
Eukaryotes have 3 different RNA
polymerases
Amanita phalloides
a-Amanitin
Sensitivity or RNA Polymerases to a-amanitin
Structure of RNA Polymerases
• All 3 are large multimeric proteins (500-700 kDa).
• All have two large subunits with sequence similarity to
b and b’ of E. coli. Therefore, catalytic sites of three
RNA Pol I, II and III may be conserved.
Epitope tagging to
Identify RNA Pol II
subunits
Subunit
structure of
yeast RNA
polymerase II
RNA Polymerase II Structure Summary
• Yeast Pol II enzyme consists of 12 different polypeptides (RPB1 - 12).
• The core subunits are RPB 1, 2 and 3.
• RPB1 and 2 are orthologs of E. coli RNA Pol b’ and
b.
• RPB1 has DNA binding site and has carboxyterminal domain (CTD). CTD has repeating
heptad PTSPSYS - a hydrophilic sequence.
• Five of the seven 7 aa’s of heptad have ‘-OH’
groups that can be phosphorylated
RNA Polymerase II Structure Summary
(cont.)
• RPB2 (b ortholgue) is at or near active site.
• RPB3/11 may function as orthologue of a-dimer of
prokaryotic RNA Pol.
• RPB5, 6, 8, 10 and 12 are shared between RNA Pol
I, II, and III.
• RPB4 and 9 are non-essential RNA Pol II subunits.
Structure of Prokaryotic and Eukaryotic RNA Pols
Eukaryotic Promoters
• Three classes of eukaryotic promoters: I, II and III which
are recognized by RNA Pol I, II, and III, respectively.
• Class I promoters not well conserved in sequence amongst
different species but general architecture consists of a
core element and an upstream promoter element (UPE).
• Some Class III promoters have promoter sequences
wholly within the gene. Another type of Class III
promoter resembles Class II promoters.
• Class II promoters have up to five conserved elements.
First consensus sequence from lining up
several eukaryotic promoters: TATA box
Generic class II promoter
TFIIB
recognition
Variable numbers
& positions
Downstream
Promoter
element
Transcription Factors
• Eukaryotic RNA Pols cannot bind to promoters on their own
(unlike prokaryotic RNA Pols).
• Transcription factors are required for eukaryotic RNA Pol
binding.
• General transcription factors allow binding of RNA Pol
binding to promoters and a basal level of transcription.
• Gene-specific factors stimulate transcription further (or
repress it) and allow fine regulatory control.
Six general transcription factors: TFIIA, B, D,
F, E and H initiate transcription.
• Gel shift assays showed that TFIID, A and B can form a
complex independently on DNA (i.e. without RNA Pol).
• TFIIF binding to DAB complex is dependent on RNA Pol.
Therefore, RNA Pol and TFIIF are needed together to join
preinitiation complex.
• Binding of TFIID to the TATA box occurs first; This event
is required for binding of all of the other TFs.
• In vitro, TFIIA is not critical for preinitiation complex
formation.
Model for DABPolF complex formation
Experimental data
TFIID
• TFIID consists of a TATA-box binding protein (TBP) and
eight TBP-associated factors (TAFIIs).
• TBP is well conserved between different organisms.
• C-terminal 180 amino acids of TBP is sufficient for
binding to the TATA box.
• TBP interacts with the minor groove of the TATA box.
This is unusual.
• TBP also functions with Pol I and III. It is a universal
transcription factor.
TBP-Associated Factors - TAFIIs
• TAFIIs attach to TBP and extend binding of TFIID
beyond TATA box in some promoters
• TAFIIs can bind initiator and downstream elements;
TAFIIs help initiate transcription from promoters
initiators and DPEs
• Specifically, TAFII150 and TAFII250 form a ternary
complex with TBP and bind to the initiator and DPE.
This was shown by crosslinking and footprinting
studies.
• In certain TATA-less promoters, TAFII250 and TAFII150
bind to initiator or DPE and recruit rest of TFIID to
promoter.
Drosphila TFIID assembled in vitro
Enhancers and Silencers
• Class II promoters have cis-acting DNA elements that
influence transcription but are not part of the promoter:
enhancers and silencers
• Trans-acting factors - proteins - act in conjunction with
cis-elements.
• Enhancers stimulate transcription while silencers inhibit
transcription. Certain elements can act as silencers or
enhancers depending on the trans-acting factors present.
• Enhancers and silencers are position and orientationindependent.
Transcription activators:
DNA binding motif
-Zinc containing modules
-Homoedomains
-bZIP and bHLH domains
Activation domain
-Acidic
-Glutamine rich
-Proline rich
Diversity of interaction with other transcription
factors and the mediator complex – could have
semester course in molecular biology on this
topic alone
Summary Picture
Also note:
Insulators
Mediator complex
Chromatin remodeling
Histone modification
Model for initiation, promoter clearance,
and elongation
CTD plays role in mRNA:
-Capping
-Splicing
-Poly-adenylation
-Termination
Eukaryotic transcription termination
by RNA Pol II
• Mechanism of termination remains unclear.
• Involves at least two elements: a polyadenlyation
signal and a downstream element (DSE) involved
in Pol II pausing.