Outline

1
Outline

• Factors for Class I promoter
• Factors for Class II promoter
• Factors for Class III promoter

2
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).
• Inducible (regulated) factors
• Work like upstream factors but are regulatory.
  Made or active only at specific times or in
  specific tissues. Interact with enhancers or
  silencers.

3
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)

4
Electrophoretic mobility shift assay (EMSA) of
TFII factors D,A,B,F on the Adenovirus major
late promoter.
Conclusions D, A, and B must form complex on DNA
for Pol II or F to bind. F must bind along with
Pol Pol cant bind w/o F.
Fig. 11.1a
5
Footprinting TFIID,A,B on a Class II Promoter
with phenanthrolineCu2 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
6
Model for forming the DABPolF complex.
(Pol extends the footprint downstream by another
34 bp total is gt 50 bp (-40 to 17).
Fig. 11.4
7
TFIID

• Binds first
• Contains TBP and 8-10 TAFIIs
• 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

8
Major Groove has more potential for recognition.
D H-bond donor H H-bond acceptor
Invert and Major Groove still different.
Fig. 9.17
9
TBP bends DNA 80o and forces open the minor
groove.
Fig. 11.6
10
TBP is required by all 3 nRNAPs!
1. Two yeast strains with temperature-sensitive
TBP were created. 2. Transcription extracts were
prepared from these strains 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.
rRNA
cyc1
Conclusion the TBP is required by all 3 RNAPs.
Fig. 11.7
11

• 2. TAFIIs of TFIID
• - 8 mostly conserved proteins ranging from 30 ?
  250 kDa

11.9 11.8
12
Functions of TAFIIs (of TFIID)

• Strongly promote transcription from promoters
  with I (initiator) and D (downstream) elements.
• X-linking (to DNA) and footprinting with
  different complexes showed that TAFII250 and
  TAFII150 bind Inr and D regions in cooperation
  with TBP.

from Fig. 11.10
13

• TAFIIs also function to
• Promote transcription from some class II
  promoters that lack a TATA box.
• Interact with some upstream activators (e.g.,
  Sp1), and hence can act as co-activators.
• Sp1 interacts with TAFII110
• Gal4 NTF-1 activator works via TAFII150 and
  TAFII60

14
TAFs are not universally required.
Based mostly on yeast strains with a
temperature-sensitive TAFII subunit. RNA from
each strain was hybridized to a microarray of
5500 yeast genes.
15
TBP is also not universally required.

• Drosophila has an alternative complex, TRF-1
  (TBP-related factor 1) , that has its own TAFs
  and promotes formation of the pre-initiation
  complex in neural tissue.
• 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.

11.17
16
TFIIA and TFIIB

• TFIIA binds to TBP and could be considered a
  TAFII
• TFIIB is needed for the Pol/TFIIF complex to bind
  to TFIID, and can be though of as a linker
  between these two.
• A current model has TFIIA binding TBP on the
  upstream side, with TFIIB binding on the
  downstream side.

17
TFIIF

• 2 subunits, called RAP70 and RAP30 (for RNAP
  associated protein).
• Binds to the RNAP, and RAP30 delivers it to the
  DAB complex.
• Reduces non-specific binding of RNAP to DNA.
• Function is analogous to the s factor
• in E. coli.

18
TFIIE and TFIIH

• TFIIE
• Binds after Pol/TFIIF binds to the pre-initiation
  complex.
• Has 2 different subunits, both needed to
  stimulate transcription.
• TFIIH
• Required for promoter clearance
• Complex protein with 9 subunits
• Has DNA helicase/ATPase activity (RAD25 gene)
• for melting DNA at transcription bubble
• Also has Kinase activity phosphorylates the
  carboxyl terminal domain (CTD) of the large
  subunit of RNAP

19
RNAP making short RNAs, its stalled at 10 - 12.
TFIIH causes further DNA unwinding, allowing the
bubble to grow and RNAP to go to elongation phase.
Fig. 11.25
20
Factors for Initiation at Class I Promoters

• Need nRNAP I and two factors, SL1 and UBF
• SL1 and UBF (a.k.a. IBF1) both bind to both
  elements (UCE Core)
• SL1 provides the species specificity
• SL1 TBP and 3 TAFIs
• TAFIs (110, 63, and 48 kDa) different from the
  TAFIIs in TFIID

21
Initiation at a Class I promoter

• UBF (IBFI UBF) binds to the UCE and core
  promoter.
• SL1 binds to UBF, and the UCE and core elements.
• RNAPI binds at core element.

22
Figure 11.34
Core
UCE
Core
23
Factors for nRNAP III

• Transcription of 5S rRNA requires 3 factors
  besides nRNAP III
• TFIIIA - binds the internal promoter, a
  Zn2- finger protein
• TFIIIC also binds to the internal promoter
• TFIIIB binds to TFIIIC, enabling TFIIIB to bind
  upstream and recruit the Pol III to the
  transcription start site

24
Effect of anti-TFIIIA on Transcription by Pol III.
Use antibody to deplete a component and test the
effect.
Figure 11.38
25
Model for the assembly of the pre-initiation
complex on a class-III tRNA promoter.
Note does not need TFIIIA.
Fig. 11.41
26
TFIIIB contains TBP 2 TAFIIIs
In vitro data indicates that TBP used by all 3
polymerases (TFIID, SL1, TFIIIB) (fig. 11.7). In
vivo evidence TBP mutant of yeast did not
synthesize 45S pre-rRNA, a mRNA or 5S rRNA.
27
Pre-initiation complexes on TATA-less promoters
contain TBP.
Assembly factor (green) binds, which promotes
binding of a TBP-complex.
Fig. 11.44
28

• The importance of TBP

TBP important for forming many transcription
initiation complexes it acts by recruiting other
proteins, either RNAPs or other general
transcription factors. The specificity of TBP for
different promoter classes resides with its TAFs.
29
Sp1 Factor for Upstream (Proximal) Class II
Promoter Element

• Binds GC boxes, stimulates transcription
• Interacts with TAFII110 in TFIID
• Also stimulates transcription of TATA-less nRNAP
  II promoter (by promoting TFIID binding)