Activating transcription
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Activating Transcription. Activation of gene structure (Chromatin structure) Initiation and processing of transcription Transport to cytoplasm Translation of mRNA. transcription, splicing of mRNA, mRNA stability, translation efficiency,.

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Activating Transcription

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Activating transcription

Activating Transcription


Activating transcription

Activation of gene structure(Chromatin structure)

Initiation and processing of transcription

Transport to cytoplasm

Translation of mRNA

transcription,

splicing of mRNA,

mRNA stability,

translation efficiency,


There are several types classes of transcription factors

There Are Several Types (Classes)of Transcription Factors

Basal factors (TFs) and RNA polymerase bind to promoter and TATAA box.

Activators are proteins that recognize specific short DNA sequences inducing the efficiency of the promoters.

Co-activators are proteins required for a more efficient transcription. They do not bind DNA.

Regulators of chromatin structure

How do co-activators and regulators work in transcription?

Figure 25.2


Independent domains bind dna and activate transcription

Independent Domains Bind DNA and Activate Transcription

  • DNA-binding activity and transcription-activation are carried by independent domains of an activator.

  • The role of the DNA-binding domain is to bring the transcription-activation domain into the vicinity of the promoter.

Figure 25.3

---DNA-binding and Activating-binding domains are independent---(TF)


The two hybrid assay detects protein protein interactions

The Two Hybrid Assay Detects Protein–Protein Interactions

  • The two hybrid assay works by requiring an interaction between two proteins:

    • one has a DNA-binding domain

    • the other has a transcription-activation domain

Figure 25.6


Activators interact with the basal apparatus

Activators Interact with the Basal Apparatus

  • The principle that governs the function of all activators is:

    • A DNA-binding domain determines specificity for the target promoter or enhancer.

  • The DNA-binding domain is responsible for localizing a transcription-activating domain in the proximity of the basal apparatus.

  • An activator that works directly has:

    • a DNA-binding domain

    • an Activating domain


Activating transcription

The two-hybrid system brings the idea of the co-activators

Activators may use a co-activator-two proteins-

Activators-one protein two domains

As long as protein complex binds both a specific DNA site and contacts the basal complex, the subunits can be encoded by separate genes.


Activating transcription

  • An activator that does not have an activating domain may work by binding a co-activator that has an activating domain.

Figure 25.7


Activating transcription

How does the activator/co-activator stimulate transcription?

1- Induce some conformational change in the Transcriptional Complex

2-Increase the binding of the RNA polymerase to the promoter

3-Increase the efficiency of the Binding/Positioning of the RNA polymerase to the promoter

How does this increase in the efficiency of the Binding/Positioning of the RNA polymerase to the promoter stimulate transcription?

  • Several factors in the basal apparatus are targets with which activators or co-activators interact.

Figure 25.8


Activating transcription

1)

2)

RNA polymerase exists as a holoenzyme (i.e. the enzyme is already associated with TFs)

Binding of the

Holoenzyme

and Mediator

Figure 25.9

TFs may manipulate the structure of chromatin


Some promoter binding proteins are repressors

Some Promoter-Binding Proteins Are Repressors

Repressor

  • Repression is usually achieved by affecting chromatin structure.

    • There are repressors that act by binding to specific promoters.

--affecting chromatin structure

--binding proteins to DNA sequence

---Expression of Histone 2B.

Testis tissues: Oct-1, CAAT

and TATA are associated

with TFs

Embryonic tissues: another

proteins (repressors), not TFs,

are associated with CAAT boxes

(CDP-CAAT-displacement

Protein)

Figure 25.10


25 7 response elements are recognized by activators

25.7 Response Elements Are Recognized by Activators

are specific DNA sequences located in both promoter /enhancer regions,

which identify genes under common regulation

Metallothionein (MT) gene

Figure 25.11

TPA (phorbol esters)

Proteins bind to these specific regulatory elements.

General principle is that any one of several elements can independently activate the gene.

What does this general principle mean?


Activating transcription

  • Each response element is recognized by a specific activator.

  • A promoter may have many response elements.

    • Elements may in turn activate transcription independently or in certain combinations.


There are many types of dna binding domains

There Are Many Types of DNA-Binding Domains

  • Activators are classified according to the type of DNA-binding specificdomain.

  • Members of the same group have sequence variations of a specific motif that confer specificity for individual target sites.


Activating transcription

Modes of regulation vs.DNA-binding domain

Transcription factors are activated in different ways

Regulation DNA-binding

Most regulatory schemes involve a modification of a protein that changes its conformation or location.

Activators may have the following domain/motifs:

Steroid receptor

Zinc finger

Helix-turn-helix

Helix-loop-helix

Leucine zipper

Sub-cellular Localization?


A zinc finger motif is a dna binding domain

A Zinc Finger Motif Is a DNA-Binding Domain

  • A zinc finger is a loop of ∼23 amino acids.

    • It protrudes from a zinc-binding site formed by His and Cys amino acids.

  • A zinc finger protein usually has multiple zinc fingers.

-(Cys, His, Phe, Leu -Zn 2+ complex)

-Cys-X2-4-Cys-X3-Phe-X5-Leu-X2-His-X3-His

-Organized as a single tandem repeats

Figure 25.13


Activating transcription

  • The C-terminal part of each finger forms an α-helix.

    • The helix binds one turn of the major groove of DNA.

  • Some zinc finger proteins bind RNA instead of, or as well as, DNA.

  • beta-sheets (?)

  • TFIIIA and elF2beta

Figure 25.14


Steroid receptors are activators

Steroid Receptors Are Activators

  • Steroid receptors are examples of ligand-responsive activators.

    • They are activated by binding a steroid (or other related molecules).

  • DNA-binding and ligand-binding domains.

Figure 25.16

Receptor-

binding

TFs-binding /

DNA-binding


Steroid receptors have zinc fingers

Steroid Receptors Have Zinc Fingers

  • The DNA binding domain of a steroid receptor is a type of zinc finger that has Cys but not His residues.

  • Glucocorticoid and estrogen receptors each have two zinc fingers.

    • The first determines the DNA target sequence.

  • Steroid receptors bind to DNA as dimers.

Figure 25.17

Steroid receptors(zinc finger) have several independent domains.Cys2/Cys2


Activating transcription

Steroid receptors fall into two groups:

Glucocorticoid (GR), mineralocorticoid (MR), androgen (AR) and progesterone (PR) receptors all form homodimers that recognize the consensus sequence TGTTCT. The estrogen receptor (ER) is a homodimer that recognizes TGACCT.

Thyroid (T3R), vitamin D (VDR), retinoic acid (RAR) and 9-cis-retinoic acid (RXR) form heterodimers and recognize the sequence TGACCT. The other part of the heterodimer is RXR.


Binding to the response element is activated by ligand binding

Binding to the Response ElementIs Activated by Ligand-Binding

  • Binding of ligand to the C-terminal domain increases the affinity of the DNA-binding domain for its specific target site in DNA.

Figure 25.19


Steroid receptors recognize response elements by a combinatorial code

  • A steroid response element consists of two short half sites that may be

  • palindromic or direct repeat.

  • A receptor recognizes its response element by the orientation and spacing

  • of the half sites

Figure 25.20

Figure 25.21

Steroid Receptors Recognize ResponseElements by a Combinatorial Code


Activating transcription

  • The sequence of the half site is recognized by the first zinc finger.

  • The second zinc finger is responsible for dimerization, which determines the distance between the subunits.


Activating transcription

without ligand

With ligand


Homeodomains bind related targets in dna

Homeodomains Bind Related Targets in DNA

  • The homeodomain (Helix-turn-Helix, HTH) is a DNA-binding domain of 60 amino acids that has three α-helices.

Figure 25.24


Activating transcription

  • The C-terminal α-helix-3 is 17 amino acids and binds in the major groove of DNA.

  • The N-terminal arm of the homeodomain projects into the minor groove of DNA.

  • Proteins containing homeodomains may be either activators or repressors of transcription.

Figure 25.25


Helix loop helix proteins interact by combinatorial association

Helix-Loop-HelixProteins Interact byCombinatorial Association

  • Helix-loop-helix (HLH) proteins have a domain of 40 to 50 amino acids.

    • The motif comprises two amphipathic α-helices of 15 to 16 residues separated by a loop.

  • The helices are responsible for dimer formation.

  • The basic regions is responsible for binding to DNA

Figure 25.26

HLH proteins have a protein association domain and a DNA binding domain.

Dimerization and basic domains are necessary for DNA binding.


Leucine zippers are involved in dimer formation

Leucine Zippers Are Involved in Dimer Formation

  • The leucine zipper is an amphipathic helix that dimerizes.

  • The zipper is adjacent to a basic region that binds DNA.


Activating transcription

  • Dimerization forms the bZIP motif:

    • The two basic regions symmetrically bind inverted repeats in DNA.

Leucine rich sequence

Dimers

Amphipathic alpha-helix

Activator Protein 1 (AP1), a transcription factor has two major subunits, Jun and Fos.

Figure 25.28


Activating transcription

RECEPTOR

Activation ?

AP1


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