This document discusses transcription factors and summarizes key details about TBP and p53. It describes transcription factors as DNA-binding proteins that recognize specific DNA sequences and form control modules that regulate gene expression. It then provides structural details of TBP, including how its saddle-shaped structure binds to the minor groove of DNA and induces bending, unwinding and widening. The document also summarizes key functions and regulatory mechanisms of the tumor suppressor protein p53, including its DNA-binding domain and oligomerization domain.
2. What is transcription factor?
Distal to the RNA Pol II initiation site, there are different combinations of
specific DNA binding sequences each of which is recognized by a
corresponding site specific DNA binding protein.
These proteins are known as transcription factor(s).
These together with DNA form the control module of gene expression
Example: TFIID,TFIIA,TFIIB, TBP etc
3. Architecture of a structural gene and the promoter(control module)
Core promoter element
4. TATA Box:
• A-T Rich 8 base pair DNA sequence
• Located 25 base pair upstream of of TSS
• Recognized by TATA Box binding Proteins (TBPs)
5. Promoter proximal Element:
• 100-200 bp long
• Several transcription factors interact directly or indirectly with the pre
initiation complex
Enhancer Element:
• Resides further upstream or down stream of the TSS
• Few thousand to 20000 bp distant from the TSS
6. Schematic model of transcriptional activation
Transcription factor Bind to the DNA
Transcriptional Activation
8. 1. TATA Box Binding Protein (TBP)
• First isolated and purified from Yeast in 1988
• Single polypeptide chain of 27 kDa
• Conserved C Terminal domain of 180 aa
• N Terminal domain of varied length and diverse sequences
• C terminal domain having DNA binding and transcription
activation function
Structure of TBP
Crystal structure by Paul Sigler @ Yale University With
Yeast C trminal TBP and Yeast TATA box DNA
Stephen Burley @ Rockyfeller University with C Terminal
TBP of A. thaliana and TATA box DNA from Adeno virus
9. • Two homologous repeat of 88 aa form
similar motifs
• Comprises of an antiparallel Beta sheet of
five strands and Two α- helices
• Two motifs are joined together by a short
loop to make a 10 stranded beta sheet
• They look like a saddle (Fig a)
• (fig b)90◦ rotation of the Fig a
10. • Loops that connects beta strand 2 & 3 of
each motif forms the Stirrups of the
saddle
• Underside of the saddle forms the
conclave surface built by the central eight
strand of beta sheet
• Side chain of this site of beta
• Sheet as well as residues of the Stirrups
forms the DNA binding Site.
• The side of the beta sheet that faces away
from the DNA is covered by two alpha
helices
Residues from these two helices and from the short loop that joins the two motif
Interacts with TFIID and with other transcription factors.
11. How TBP binds to the DNA?
Answer: TBP binds to the minor groove of the DNA
and Induces large structural changes
12. • Normal B-DNA structure returns out side the TATA box
• The helical axis of the DNA at each end of the TATA BOX
form an angle of about 100 degree to each other , instead of the
Expected 180 degree if the DNA was not bent.
• First two and the last two bp of TATA box, there are sharp kinks, DNA is
Covered smoothly and partially unwounded.
13. • Two Phenylalanine residues are partially inserted between first two and the last two bases, preventing
stacking of the adjacent bases and allow increase in rise Of the DNA
• The kinks at each end of the DNA and partial unwinding of the DNA produces a wide and shallow minor
groove.
• This exposed wide and shallow minor groove bind intimately to the concave undersurface of the TBP saddle.
DNA Modifications: Distortions:
a. Bending of DNA
b. Widening of the minor groove
c. Unwinding of the DNA
14. • All eight nucleotides of TATA box interacts with TBP and their structure
deviates from the normal B-DNA.
• Saddle would straddle normal B-DNA structure with helical axis of the DNA
perpendicular to a line connecting the two stirrups.
• DNA is sharply bent at TATA box region so that the local helical axis is
almost is almost parallel to the line from stirrups to stirrups.
Protein
Saddle structure
Minor groove of
DNA
15. What is the nature of the interaction?
• Strong hydrophobic interaction between the underside of TBP saddle and the minor
groove of DNA
• Side chains of eight central beta strands interacts with both the phosphate sugar Backbone
and the minor groove of the eight nucleotides of the TATA box.
• Fifteen side chains projecting from the beta strands make hydrophobic contacts With the
sugar and bases of DNA.
• The phosphate groups are hydrogen bonded to arginine and lysine side chains At the edges
of the interaction area.
16. Why specific to TA/AT sequence at 4 and 5 position of
bp?
Only sequence specific H bonds – center of box
Asn 69 – O2 of T4’ and N3 of A5’
Asn 159 – O2 of T5 and N3 of A4
Thr 124 &215– N3 of A both sides
Role of Conserved Val residues.
Val 71 and 122 on one side
Val 161 and 213 on the other side
Side chains of Val residues cause steric interference with NH2 substituent from
G-C or C-G basepair.
Flanking Val residues in combination with 6 H-bonds specify A-T or T-A at
positions 4 and 5 of TATA box
Why Minor groove???
Quasi – palindromicity
Functional implication of DNA bending
TBF – associated factors (TAF)
Significance of N – terminal in TBP
17. Why strong affinity between TBP-TATA Box? Around 100000 fold
more affinity than random DNA.
• Large interacting hydrophobic surface area
• Major distortion in the DNA
• SIX Hydrogen bonds between 4 side chain residues of TBP and 4
hydrogen bond acceptors from bases In the minor groove.
18. 2. p53
Most ambiguous and cited biological molecule.
Encoded by genes known to be Tumor Suppressor Genes (TSG)????
Protein with 53 kDa MW – promotes expression of p21 – a protein inhibiting
CDK’s (Check point) in the cell cycle-
This gives sufficient time to repair or destruction of damaged cells (apoptosis)
Single point mutation – altered function – observed in more than half of the cancer
patients
wild type – sequence specific DNA binding
mutated p53 – no binding and hence no regulation- leads to no expression of
p21 and hence uncontrolled cell cycle.
19. p53 – Oligomerization Domain
Oligomerization domain – tetramer formation of p53
Mutations in C – terminal affects tetramer formation.
The monomer still retains DNA binding function
No complete structure available
available structures- Sloane Kettering institute for cancer- NewYork
21 base pairs sequence bound to p53 DNA binding region (102 – 292)
Oligomerizing domain (325 – 356)
Each unit of p53 has a beta strand –turn- alpha helix
Two units bind together by antiparallel beta sheet- followed by antiparallel helix
formation
This dimer binds with another dimer by hydrophobic interactions of the helix. Beta
sheets do not interact in the tetramer
Tumorogenic mutations
Leu 330 to His- water loving His does not allow dimerization core to be formed
Glycine in turn if mutated to any other residue - also abolishes p53 dimerization
20. P53 – DNA binding Domain
DNA binding domain (anti-parallel beta barrel)
protruding loops from anti – parallel beta barrel
immunoglobulin fold (7/9 strands)
This kind of fold also present at I– MHC binding coreceptor in CD4
NF-kB – REL homology region
