Transcript Lec. 14 - Utexas

The SV40 Promoter
Enhancer
GC boxes (6)
TATA box
Transcription Factors for Class II
promoters (RNAP II)
• Basal factors
Required for initiation at nearly all promoters;
determine site of initiation; interact with TATA box.
• Upstream factors
DNA binding proteins that recognize consensus
elements upstream of TATA box. Ubiquitous. Increase
efficiency of initiation. Interact with proximal promoter
elements (e.g., CCAAT box).
• Regulated factors
Work like upstream factors but are regulatory. Made
or active only at specific times or in specific tissues.
Interact with enhancers, silencers, or insulators.
Basal Factors Required to form the
Class II Pre-Initiation Complex
• 6 factors + RNAP II = Pre-Init. Complex
• Factors :
– TFIIA, TFIIB, TFIID, TFIIE, TFIIF,
TFIIH
– Many are multi-subunit
– Factors + RNAP must also bind in a
specific order (in vitro)
Electrophoretic
mobility shift
assay (EMSA) of
TFII factors
D,A,B,F on the
Adenovirus major
late promoter.
Conclusions:
1. D and B must form a
complex on DNA for Pol II
or F to bind.
2. F must bind along with
Pol; Pol can’t bind w/o F.
Fig. 11.1a
Footprinting TFIID,A,B
on a Class II Promoter
with phenanthroline–
Cu2+ and with DNAse I.
D makes footprint,
which is enhanced by
A.
B does not expand the
footprint, but makes
+10 nt more reactive.
Notice that DNase I
gives a bigger footprint
than OP-Cu2+.
Fig.
11.2b
Model for forming the DABPolF complex.
(Pol extends the footprint downstream by another 34 bp:
total is > 50 bp (-40 to +17).
Fig 11.4
TFIID
•
•
Binds first
Contains TBP and 8-10 TAFIIs
1. TBP = TATA box binding protein
- 38 kDa protein
- Highly conserved C-terminal domain for
binding TATA box
- Saddle-like structure
- Binds DNA via the minor groove
Major Groove
has more
potential for
recognition.
D= H-bond donor
A= H-bond acceptor
Invert and Major
Groove still
different.
Fig 9.14
TBP bends
DNA ~80o and
forces open the
minor groove.
Similar to Fig. 11.6
TBP is required by
all 3 nRNAPs!
1. A yeast strain with a
temperature-sensitive TBP was
created.
2. Transcription extracts were
prepared from this strain and
from wild-type (WT).
3. The extracts were treated at
the indicated temperature, and
then used to transcribe a gene
for each RNAP.
4. Transcription was assayed
by S1 mapping the 5’ end of
each RNA.
Conclusion: the TBP is
required by all 3 RNAPs.
Fig. 11.7
rRNA
cyc1
2. TAFIIs of TFIID:
~8 mostly conserved proteins
ranging from 30  250 kDa
11.9
11.8
Functions of TAFIIs
(TFIID):
1. Strongly promote
transcription from promoters
with I (initiator) and D
(downstream) elements,
such as Hsp70 in
Drosophila (right).
2. X-linking (to DNA) and
footprinting with different
complexes showed that
TAFII250 and TAFII150 bind
I and D regions in
cooperation with TBP.
from Fig. 11.10
Footprinting TBP and a
Ternary TAF Complex
on the HSP70 Promoter
Conclusion: The Inr and DPE
regions are bound by TAFII250
and/or TAFII150
Fig 11.12
TAFIIs also function to:
1. Promote transcription from some class II
promoters that lack a TATA box.
2. Interact with some upstream activators
(e.g., Sp1), and hence can act as coactivators.
• Sp1 interacts with TAFII110
• Gal4 NTF-1 activator works via TAFII150
and TAFII60
TAFs are not universally required.
Based mostly on yeast strains with a temperaturesensitive TAFII subunit. RNA from each strain was
hybridized to a microarray of 5500 yeast genes.
TBP is also not universally required.
• Drosophila has an alternative complex, TRF-1 (TBPrelated factor 1), that his its own TAFs (nTAFs) and
promotes formation of the pre-initiation complex in
neural tissue (binds TFIIA and TFIIB).
• The Drosophla tudor gene has two promoters, one with
a TATA box that binds TBP, and the other a TC box
that binds TRF-1.
11.15
TFIIB (and TFIIA)
• TFIIB is
needed for the
Pol/TFIIF
complex to
bind to TFIID
TFIIB-TBP-Pol IIDNA structure
• TFIIA binds to
TBP and could
be considered
a TAFII
TFIIF
• 2 subunits, called RAP70 and RAP30
(for RNAP associated protein).
• Binds to the RNAP; RAP30 delivers it
to the DAB complex.
• Reduces non-specific binding of the
RNAP to DNA, analogous to the s
factor in E. coli.
TFIIE and TFIIH
•
1.
2.
TFIIE
Binds after Pol/TFIIF binds
Stimulates TFIIH
•
1.
TFIIH
Also required for promoter
clearance
Complex (9 subunits)
DNA helicase activity (for
melting DNA)
Kinase activity phosphorylates the CTD of
the large subunit of RNAP
2.
3.
4.
Fig 11.20
RNAP is
stalled
at +10 - +12.
Fig. 11.25
TFIIH (+ATP) further
unwinds DNA, expanding
the bubble and allowing
RNAP to go to elongation
phase.
TEFb further
phosphorylates the
CTD.
Model for the Class II Preinitiaiton Complex
Elongation Factor TFIIS and Proofreading
11.28
11.27
TFIIS
- stimulates elongation by limiting pauses
- stimulates proofreading by promoting the
RNase activity of the RNAP II
11.28
Class I Promoters (for nRNAP I)
SL1
UBF
-150
-100
UCE
-50
+20
Core
- SL1 contains TBP and 3 TAFIs
- the human complex
Factors for Initiation at Class I
Promoters
• nRNAP I needs two factors, SL1 and UBF
1. SL1 binds to the Core element, and
provides species specificity
- composed of TBP plus 3 TAFIs
- the TAFIs are 110, 63, and 48 (different
from the TAFs in TFIID)
2. UBF binds UCE (UPE), acts synergistically
by promoting SL1 binding to Core
Factors for nRNAP III
•
Transcription of all class III
genes requires TFIIIB and
TFIIIC, but TFIIIA needed for
transcription of 5S rRNA
1. TFIIIC - binds the internal
promoter
2. TFIIIB - binds to TFIIIC and
upstream DNA, recruits the
RNAP
3. TFIIIA - binds internal
promoter, a Zn2+-finger
protein
Model for the
assembly of
the
preinitiation
complex on a
class III tRNA
promoter
Note: does not
need TFIIIA.
Fig. 11.39
TFIIIB contains TBP + 2 TAFIIIs
In vitro data indicates that TBP is used by all 3
polymerases (TFIID, SL1, TFIIIB).
In vivo, the TBP mutant of yeast did not
synthesize 45S pre-rRNA, a mRNA, or
5S rRNA.
Pre-initiation
complexes on
TATA-less
promoters for all
3 RNAPs.
Assembly factor
(green) binds, which
promotes binding of a
TBP-complex.
Fig 11.42
The importance of TBP
TBP is important for forming many transcription
initiation complexes. It acts by recruiting other
proteins, either the RNAP or other general
transcription factors (class II). The specificity of
TBP for TATA-less promoters – i.e., what kind of
promoter it will activate, resides with its TAFs.