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CHAPTER 28 (Genes X)

EUKARYOTIC TRANSCRIPTION

REGULATION

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Control of Gene Expression in EukaryotesEukaryotic gene expression is usually controlled at the

level of initiation of transcription.

Control of Gene Expression in EukaryotesEukaryotic gene expression is usually controlled at the

level of initiation of transcription.

GC boxGC boxsilencerssilencers

enhancersenhancers

CCAAT box

(called the CAT box)

CCAAT box

(called the CAT box)

the TATA boxthe TATA box

the promoterthe promoter

Methylation

Hormonal Control

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Thinking about Gene Regulation

Humans begin life from a single cell; all the genetic information needed to create an adult is in our genome.

Embryonic cells undergo differentiation to produce specific cell types such as muscle, nerve, and blood cells.

Different cell types are the consequence of differential gene expression.

Humans begin life from a single cell; all the genetic information needed to create an adult is in our genome.

Embryonic cells undergo differentiation to produce specific cell types such as muscle, nerve, and blood cells.

Different cell types are the consequence of differential gene expression.

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A typical differentiated mammalian cell makes about 10,000 proteins from approximately 30,000 genes.

Most of these are housekeeping proteins needed to maintain all cell types.

Certain proteins can only be detected in specific cell types.

How is gene expression regulated?

A typical differentiated mammalian cell makes about 10,000 proteins from approximately 30,000 genes.

Most of these are housekeeping proteins needed to maintain all cell types.

Certain proteins can only be detected in specific cell types.

How is gene expression regulated?

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COORDINATELY CONTROLLED GENES

• PROCARYOTES– Clustered in operons

– Same regulatory sites

– Single mRNA molecule

– Translated together

• EUCARYOTES– NO operons !

– Genes individually transcribed

– Own promoter

– Specific enhancers for each gene

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(B) In procaryotes the production of mRNA molecules is much simpler. The 5' end of an mRNA molecule is produced by the initiation of transcription by RNA polymerase, and the 3' end is produced by the termination of transcription. Since procaryotic cells lack a nucleus, transcription and translation take place in a common compartment. In fact, translation of a bacterial mRNA often begins before its synthesis has been completed.

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Genes have regulatory DNA sequences upstream from the initiation site where transcription begins.

The promoter is the RNA polymerase binding site.

Gene regulatory proteins bind to regulatory DNA sequences and can either prevent or enhance RNA polymerase binding.

Co-activators

Activators in enhancer

Activators in distal promoter

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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.

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Acidic activators: They have multiple negative charges

Yeast activators: GAL4 , GCN4

Herpes Simplex Virus: VP16

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The modular structure of a gene activator protein. Outline of an experiment that reveals the presence of independent DNA-binding and transcription-activating domains in the yeast gene activator protein Gal4. (A) The normal activation of gene transcription produced by the Gal4 protein. (B) The chimeric gene regulatory protein requires the LexA protein DNA-binding site for its activity.

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Identification of Protein-Protein Interactions by the Yeast 2-Hybrid

System

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The two hybrid techniques tests the ability of two proteins to interact by incorporating then into hybrid proteins where one has a DNA-binding domain and the other has a transcription-activating domain.

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Yeast Gal4

Transcription factor• Zinc Finger DNA binding domain (DBD)• Transcriptional Activation Domain (AD)

ADDBD

Gene XON

promoter

Yeast Two Hybrid Assay

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Yeast Two Hybrid Assay

DBD

Bait

The bait: cDNA of the protein you want to find partners for.

Reporter GeneOFF

promoter

AD Prey

The prey: from random cDNA’s whichencodes for various proteins. Do any interact with the bait?

Reporter GeneOFF

promoter

Reporter GeneON

promoterYeast cells turn blue

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Yeast Two Hybrid Assay

Uetz, 2001

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Activators interact with the basal apparatus

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

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

•An activator that works directly has a DNA-binding domain and an activating domain.

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

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

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An activator may bind a co-activator that contacts the basal apparatus.

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Activators may work at different stages of initiation, by contacting the TAFs of TFIID or contacting TFIIB.

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How does an activator stimulate transcription?

Two models:

1. The recruitment model: Activators sole effect

is to increase the concentration of RNA

polymerases to the promoter.

2. Alternative model: Activator induces some

change in the transcriptional complex, i.e.

conformational change in enzyme, increases its

efficiency.

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RNA polymerase may be associated with various alternative sets of transcription factors in the form of a holoenzyme complex.

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A model for the action of some eukaryotic transcriptional activators. The gene activator protein, bound to DNA in the rough vicinity of the promoter, facilitates the assembly of some of the general transcription factors. Although some activator proteins may be dedicated to particular steps in the pathway for transcription initiation many seem to be capable of acting at several steps.

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Some Promoter Binding Proteins are Repressors

• Repression is usually achieved by affecting

chromatin structure, but there are repressors that act

by binding to specific promoters and function like

trans-acting (such as bacterial repressors) to block

transcription.

• The global repressor NC2/Dr1/DRAP1, is a

heterodimer that binds to TBP to prevent it from

interacting with other components of the basal

apparatus.

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A transcription complex involves recognition of several elements in the sea urchin H2B promoter in testis. Binding of the CAAT displacement factor (CDP) in embryo prevents the CAAT-binding factor from binding, so an active complex cannot form.

In embryo

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Take away message:

The function of a protein in binding to a known promoter element

cannot be assumed: It may be activator, a repressor, or even

irrelevant to gene transcription.

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Transcription factors are activated in several ways

The activators are classified according to their The activity of an inducible activator may be regulated by several ways

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Oncogenes that code for transcription factors have mutations that inactivate transcription (v-erbA and possibly v-rel) or that activate transcription (v-jun and v-fos).

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Transcription is controlled by proteins binding to regulatory DNA sequences.

Promotor includes

RNA polymerase binding site

initiation site

Regulatory DNA sequences bound by gene regulatory proteins

some short – simple gene switches

some long and complex (eukaryotic)

molecular microprocessors which respond to a variety of signals, integrate them, and determine the rate of transcription.

Expression depends on;

cell type

its environment

its age

extracellular signals

Edge of bases

Gene Regulatory Proteins Bind to DNA

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Gene regulatory proteins insert into the major groove,

contacts and binds (non-covalently) to the edges of the

bases (about 20 interaction), usually without disrupting

the hydrogen bonds.

This binding is very strong and very specific for the

nucleotide sequence. Frequently DNA-binding proteins

contain alpha-helices bind in pairs - dimerization - which

doubles the contact area, increasing the strength and

specificity of the interaction.

Gene Regulatory Proteins Bind to DNA

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There are many types of DNA binding domains

The activators are classified according to their DNA binding domain.

• Zinc Finger• Steroid Receptors• Helix-turn-helix motif• Homeodomain• Helix-loop-helix• Leucine zippers

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

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DNA binding zinc-finger motifs

One or more zinc atoms are added to the structure.

1. It is found in in a cluster with additional zinc fingers, arranged one after another, the helix of each contact the major groove of the DNA, foring a continous strech of a helices along the groove.

2. Two a helices are packed together with zinc atoms. Mostly found in the large family of intracellular receptor proteins (dimers).

• Cys2/His2 Finger

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

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Transcription factor SP1 has a series of three zinc fingers each with a characteristic pattern of cysteine and histidine residues that constitute the zinc-binding site.

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Zinc fingers may form a-helices that insert into the major groove, associated with b-sheets on the other site.

Cys2/His2 finger

7-8 aa

~23 aa

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DNA binding by a zinc finger motif: The zinc-finger motif is composed of an alpha helix and a beta sheet.Each zinc-finger is held together by a molecule of zinc.

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A dimer of the zinc finger domain of the intracellular receptor family bound bound to its specific DNA sequence. (e.g., glucocorticoid receptor)

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-sheet can also recognized DNA: i.e. bacterial met repressor. It is a dimeric protein. Each dimers sheet can bind to the major goorve of the DNA.

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DNA binding by a zinc finger protein: This protein recognizes DNA using three zinc fingers of the cys-cys-his-his type arranged as direct repeats.

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Sequence specific interactions between different six fingers and their DNA recognition sequences.

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Steroid receptors have several independent domains

All the receptors have independent domains for DNA binding and hormone binding.

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Steroid receptors have zinc fingers

The first finger of a steroid receptor controls which DNA sequence is bound (red);The second finger controls spacing between the sequences (blue).

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Discrimination between GRE and ERE target sequences is determined by two amino acids at the base of the first zinc finger in the receptor.

Cys-X2-Cys-X13-Cys-X2-Cys

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Each receptor recognizes response elements that are related to a consensus.

• These response elements have a characteristic feature:Each consensus consists of two short repeats (or half sites). The receptors bind as a multimer, so that each half of the consensus is contacted by one subunit.

• The response elements for the various receptors may be either palindromes or direct repeats in which the half sites are separated by 0-4 bp whose sequence is irrelevant.

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Response elements formed from the palindromic half site TGTTCT are recognized by several different receptors depending on the spacing between the half sites.

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The receptors fall into two groups:

1) GR, MR, AR, PR receptors all form homodimers. They recognize half sites, TGTTCT consensus sequence, arranged as palindormes. The spacing between the elements determins the type of element.

2) T3R, VDR, RAR, 9-cis retinoic acid (RXR) receptors form heterodimers, which recognize half elements with the sequence TGACCT.

1bp - RXR - RXR3bp - VDR - RXR4bp - T3R - RXR5bp - RAR - RXR

They can also form homodimers, which recognize palindromes.

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Response elements with direct repeats TGACCT are recognized by heterodimers of which one member is RXR.

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Homeodomain proteins:

Homeotic selector genes play a critical role in

Drosophila development. Several homeotic selector genes contain almost

identical streches of 60 amino acids that defines this

class of proteins called “homeodomain”. Homeodomain proteins are present virtually all

eucaryotic organisms. The heli-turn-helix motif in homeodomains is always

surrounded by the same structure suggesting that the

motif is always presented to DNA in the same way.

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A homeodomain bound to its specific DNA sequence.In the homeodomain, three alpha helices interact with the DNA. Helix 2 and 3 closely resembles the helix-turn-helix motif.Asparagine in helix number 3 binds to adenine in the DNA

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Comparison of homeodomains from two organisms separated by more than a billion years of evolution. The Yeast 2 protein (green) and Drosophila engrailed protein (red). Black dots mark sites with identical amino acids. Orange dots indicate the position of a three amino acid insert in the 2 protein.

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Helix-loop-helix proteins interact by combinatorial association

Helix-loop-helix proteins have a motif of 40-50 amino

acids that comprises two amphipathic -helices of 15-16

residues separated by a loop. The helices are responsible for dimer formation.

bHLH proteins have a basic sequence adjacent to the

HLH motif that is responsible for binding to DNA. Class A bHLH proteins are ubiquitously expressed.

Class B bHLH proteins are tissue specific. A class B protein usually forms a heterodimer with a

class A protein. HLH proteins that lack the basic region prevent a bHLH

partner in a heterodimer from binding to DNA.

HLH proteins form combinatorial associations that may be changed during development by the addition or removal of specific proteins

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A helix-loop-helix dimer bound to DNA. The two monomers are held together in a four-helix bundle.: each monomer contributes two a helices connected by a felixible loop of protein (red). A specific DNA sequence is bound by the two a helices that are project from the four-helix bundle.

HLH can make both homo and heterodimers

Flexible loop

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An inhibitory regulation by truncated HLH proteins.HLH motif is responsible both dimerization and DNA binding.It recognizes a symmetric DNA sequence.

Lacks DNA binding domain

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Formation of muscle cells is triggered by several bHLH proteins, including MyoD as a heterodimer MyoD-E12 or MyoD-E47, rather than a MyoD homodimer. Before myogenesis begins, the Id protein may bind to MyoD and or E12 or E47 to form hterodimers that can not bind to DNA. So removal of Id could be the trigger that releases MyoD to initiate myogenesis.

An HLH dimer in which both subunits are of the bHLH type can bind DNA, but a dimer in which one subunit lacks the basic region cannot bind.

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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.

Dimerization forms the bZIP motif in which the

two basic regions symmetrically bind inverted

repeats in DNA.

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In the leucine zipper motif, two alpha helices interact with each other to allow the DNA the DNA binding domain of their protein to bind to DNA.The two alpha helices may be from different proteins.

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Heterodimerization of leucine zipper proteins can alter their DNA-binding soecificity.Two different monomers can combine to form a heterodimer, which now recognizesa hybrid DNA sequence.

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Types of DNA binding proteins

DNA and RNA polymeraserepair enzymes

structural proteinstranscription factors

Types of DNA binding proteins

DNA and RNA polymeraserepair enzymes

structural proteinstranscription factors

DNA binding motifs

zinc fingersleucine zippershelix-turn-helixhelix-loop-helix

DNA binding motifs

zinc fingersleucine zippershelix-turn-helixhelix-loop-helix

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It is not yet possible to accurately predict the DNA sequences recognized by all gene regulatory proteins.

Is there a simple amino acid-base pair recognition code: e.g., is a G-C base pair always contacted by a particular amino acid side chain?

Although certain amino acid-base interactions appear much frequently than others the answer is NO…

One of the most common protein-DNA interactions

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Protein surface virtually any shape and chemistry

can be made from just 20 different amino acids, and

a gene regulatory protein uses different

combinations of these to create a surface that is

precisely complementary to a particular DNA

sequence. We know that the same base pair can be

thereby be recognized in many ways depending on

its context.

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Sequence specific interactions between different six fingers and their DNA recognition sequences.

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HOW GENETIC SWITCHES WORK?

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An operon = a cluster of bacterial or viral genes transcribed from a single promoter.

A sequence of DNA within the promotor is recognized by regulatory proteins - the operator.

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Repressor protein switch genes on or off

Is recognized well by RNA polymerase.

Bacteria in your gut after you eat a steak

Regulatory proteins like these are allosteric.

These regulators allow fast response to the environment because they are always present in the cell - constitutively expressed

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The binding of tryptophan to the tryptophan repressor protein changes the conformation of the repressor. It is an helix-turn-helix protein.

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Addition of an “inducing” ligand can turn on a gene either by removing a gene repressor protein from the DNA or by causing a gene activator protein to bind.

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Activator proteins act on promoters that do not bind RNA polymerase on their own. These promoters are made fully functional by the addition of a bound activator protein which is also allosteric, activated by binding another molecule at a site different from the DNA binding site.

Example: CAP has to bind cyclic AMP (an intracellular signaling molecule) to bind DNA. Genes regulated by CAP are switched on in response to increases in cAMP, which is triggered by signals received by cell membrane receptors.

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A model for gene activation from an enhancer

General transcription factors usually can not efficiently initiate transcription alone.

Enhancers can be thousands of base pairs away, either upstream or downstream and can either increase or decrease transcription.

“Action at a distance”

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Summary of gene activation and regulation in procaryotes and eucaryotes.

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The gene control region of a typical eucaryotic gene.

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Gene Regulatory sequences of a typical eucaryotic cell

Regulated by combinations of proteins over up to 50,000 nucleotide pairs

Combinatorial control can be positive or negative