TATA-Binding Protein Mutants

Familiarize yourself with the main list of calculations.

Step-by-step approaches in checking the correctness of building X-ray structures (PDB files)

Biomarker development concept

Calculation of biochemical pathways of biochemical reactions taking into account oncogenic mutations and gene expression.

Cryo-EM structures of TFIID and its binding to DNA.

1. Recent insights into the structure of TFIID, its assembly, and its binding to core promoter// Published in final edited form as:

Curr Opin Struct Biol. 2020 April ; 61: 17–24. doi:10.1016/j.sbi.2019.10.001.

Symbol: GTF2F2
Name: general transcription factor IIF, polypeptide 2, 30kDa
Synonyms: BTF4
RAP30
TFIIF

Symbol GTF2B
Name general transcription factor IIB
Synonyms TFIIB

Symbol: GTF2A2
Name: general transcription factor IIA, 2, 12kDa
Synonyms: HsT18745
TFIIA

Symbol: TBP
Name: TATA box binding protein
Synonyms: GTF2D1
SCA17
TFIID

Structure

Structure

Structure

Structure

A

Initial stage.
Comparison of calculated and experimental data.

the calculated stability value lg(cond(W)) is qualitatively proportional to the experimental value Kd

Verification of calculated and experimental data before conducting a large-scale computational process

An additional controlled parameter is the measure of change in the differential entropy (delta)H

The effect of the R188E is a direct effect on DNA binding by the TBP_CORE

Figure 1. Wild-Type and Activated DNA Binding Mutant TBP Molecules Have Different TATA Box On Rates and Different DNA Binding Surfaces. Electrophoretic mobility retardation analyses of the rates of formation of the TBP-TATA box complex on the U6 promoter with (A) wild-type TBP and the (B) TBPR188E mutant TBP molecules.
A master binding reaction was prepared at 30C, and aliquots were removed at 1, 4, 9, 16, 25, 36, 49, 64, 81, and 100 min and immediately loaded onto the gel.

After incubation of a master binding reaction for 100 min at 30C, excess unlabeled DNA was added, and aliquots werremoved after an additional 1, 4, 9, 16, 25, 36, 49, 64, 81, and 100 min incubation at 30C and immediately loaded onto the gel. The graphs below the autoradiograms show the dissociation of TBP from the TBP-TATA box complexes over incubation time after addition of the competitor DNA. The unit of the vertical axis is arbitrary. Filled dots and open triangles represent the unbent TBPFL and bent TBPFL* complexes respectively.

Figure 2. The Bent, But Not the Unbent, Wild-Type TBP-TATA Box Complex Is Very Stable. Electrophoretic mobility retardation analysis of the dissociation rates of (A) TBPWT and (B) TBPR188E molecules from the TBP-TATA box complexes on the U6 promoter.

TBP_R188E formed a TBPFL* complex rapidly beginning within 1 min of incubation and formation of the complex increased rapidly with timenot simpler.

C

lg(cond(W))_______Kd

neither alanine nor radical
mutations showed any activated DNA binding activity.[1]

radical mutations showed activated TBPFL* DNA binding, although the radical mutations were more active.[1]

the radical but not alanine mutations activated formation of
the TBPFL* complex[1]

1. A Regulated Two-Step Mechanism of TBP Binding to DNA: A Solvent-Exposed Surface of TBP Inhibits TATA Box Recognition

Cell, Vol. 108, 615–627, March 8, 2002, Copyright 2002 by Cell Press
Xuemei Zhao and Winship Herr
Cold Spring Harbor Laboratory
Cold Spring Harbor, New York 11724

In the standard 30 min incubation, residues are either inactive or only form the TBPFL complex for both alanine and radical substitutions: L185(A and K), E191(K), R203(A and E), E206(A and K), R208(A and E)[1]

D

decrease in stability
DNA-TBP binding

increased in stability

increased in stability

increased in stability
DNA-TBP binding

decrease in stability

Symbol: TBP
Name: TATA box binding protein
Synonyms: GTF2D1
SCA17
TFIID

Symbol: GTF2B
Name: general transcription factor IIB
Synonyms: TFIIB

Symbol: GTF2A2
Name: general transcription factor IIA, 2,
Synonyms: HsT18745
TFIIA

Ниже приведены описание и перечень таких мутаций для выбранных белков TBP, GTF2B, GTF2A, для которых будет выполнен расчет связывания c учетом различных мутаций в белках, взятых из Cancer Genome Atlas.

Determine the effect of each of the mutations on the course of biochemical reactions.

E

Symbol: GTF2B
Name: general transcription factor IIB
Synonyms: TFIIB.

Each molecular complex is characterized by its stability value lg(cond(W)), while the formation of each complex is characterized by its forward and reverse chemical reaction constants Kn and K-n

TFIIB

4.6375_________ _____5.265e-19___________ 1.213e-23______ 6.266e-32

__number ___________________ max _________________min _________________error condition__________________________________________________________computational SVD

F

__number ___________________ max _________________min _________________error condition__________________________________________________________computational SVD

4.681____________________5.929e-19_________1.235e-23___________5.191e-19

[TFIIB-TBP-DNA]

DNA

TBP

__number ___________________ max _________________min _________________error condition__________________________________________________________computational SVD

2.643____________5.1678e-19______________1.174e-21____________6.478e-32

Symbol: TBP
Name: TATA box binding protein
Synonyms: GTF2D1
SCA17

Figure 1. Structure of CH3-GTF2A2 dimer

Figure 2. Structure of biological complex
[TBP-DNA-TFIIB-THIIA-CH3]

Figure 3.Calculation results for various structures in which various substitutions of amino acid residues were introduced.

Figure 4. Calculation results for various structures in which various substitutions of amino acid residues were introduced.

Calculation results for various dimer structures and four proteins associated with DNA, in which various amino acid residue substitutions were introduced.

G

CH3

GTF2A2