Transcription Factors for Class II promoters RNAP II

1
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.

2
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)

3
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 cant bind w/o F.
Fig. 11.1a
4
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
5
Model for forming the DABPolF complex.
(Pol extends the footprint downstream by another
34 bp total is gt 50 bp (-40 to 17).
Fig. 11.5
6
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

7
Major Groove has more potential for recognition.
D H-bond donor A H-bond acceptor
Invert and Major Groove still different.
Fig. 9.17c
8
TBP bends DNA 80o and forces open the minor
groove.
Similar to Fig. 11.6
9
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.
rRNA
cyc1
Conclusion the TBP is required by all 3 RNAPs.
Fig. 11.7
10

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

11.9
11.8
11
Functions of TAFIIs (TFIID)

• Strongly promote transcription from promoters
  with I (initiator) and D (downstream) elements,
  such as Hsp70 in Drosophila (right).
• 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. 11.10a
12

• 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

13
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.
14
TBP is also not universally required.

• Drosophila has an alternative complex, TRF-1
  (TBP-related 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.

11.17
15
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 thought 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.

16
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.

17
TFIIE and TFIIH (needed for promoter clearance)

• TFIIE
• Binds after Pol/TFIIF binds
• 2 different subunits, both are needed
• Stimulates TFIIH
• TFIIH
• Required for promoter clearance
• Complex protein, 9 subunits
• DNA helicase activity (RAD25 gene), for melting
  DNA at transcription bubble
• Kinase activity - phosphorylates the CTD of the
  large subunit of RNAP

18
RNAP is stalled at 10 - 12.
TFIIH (ATP) further unwinds DNA, expanding the
bubble and allowing RNAP to go to elongation
phase.
TEFb further phosphory-lates the CTD.
Fig. 11.25
19
Elongation Factor

• TFIIS
• stimulates elongation by limiting pauses
• stimulates proofreading (by promoting the RNase
  activity of the RNAP II)

20
Factors for Initiation at Class I Promoters

• nRNAP I needs two factors, SL1 and UBF
• 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

21
Class I Promoters (for nRNAP I)
SL1
UBF
-150 -100 -50 20
UCE
Core
- SL1 contains TBP and 3 TAFIs
22
Factors for nRNAP III

• Transcription of all class III genes requires
  TFIIIB and TFIIIC
• Transcription of 5S rRNA also requires TFIIIA
• TFIIIC - binds the internal promoter
• TFIIIA - binds internal promoter, a Zn2 -finger
  protein
• TFIIIB - binds to TFIIIC and upstream DNA,
  recruits the RNAP to transcrip. start site.

23
Model for the assembly of the pre-initiation
complex on a class-III tRNA promoter.
Note does not need TFIIIA.
Fig. 11.41
24
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.
25
Pre-initiation complexes on TATA-less promoters
contain TBP.
Assembly factor (green) binds, which promotes
binding of a TBP-complex.
Similar to Fig 11.44
26

• The importance of TBP

TBP 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.
27
The Sanger or dideoxy method of DNA sequencing.
1. 4 replication reactions, same primer but each
with a limited quantity of a different
dideoxynucleotide (ddNTP). 2. Incorporation of
the ddNTP terminates the reaction. 3. The 4
reactions are electrophoresed side-by-side on a
denaturing polyacrylamide gel. 4. Sequence is
read from bottom to top.
Figs 5.18, 5.19
28
Automated DNA Sequencing
M. Hunkapillar L. Hood
1. The dideoxynucleotides are tagged with a
fluorescent group. 2. Each type of dideoxy
fluoresced a different color. 3. Only 1 reaction
needed, and only 1 lane of a gel to sequence a
stretch of DNA. 4. As DNA fragments reach the
bottom of the gel, a laser excites the dideoxy
and sensors detect the fluoroscence. 5. A
computer converts it to a linear sequence.