Molecular BiologyFifth Edition

Chapter 12

Transcription Activators in Eukaryotes

Lecture PowerPoint to accompany

Robert F. Weaver

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

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Transcription Activators of Eukaryotes

• The general transcription factors by themselves dictate the starting point and direction of transcription but they are capable of sponsoring only a low level of transcription or basal transcription

• Transcription of active genes in cells rises above the basal level

• Eukaryotic cells have additional, gene-specific transcription factors called activators that bind to DNA elements called enhancers to provide the extra needed boost to transcription

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12.1 Categories of Activators

• Activators can stimulate or inhibit transcription by RNA polymerase II

• Structure is composed of at least 2 functional domains– DNA-binding domain– Transcription-activation domain– Many also have a dimerization domain

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DNA-Binding Domains

• Protein domain is an independently folded region of a protein

• DNA-binding domains have DNA-binding motif– Part of the domain having characteristic

shape specialized for specific DNA binding– Most DNA-binding motifs fall into 3 classes;

zinc-containing modules, homeodomains and bZIP and bHLH motifs

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Zinc-Containing Modules

• There are at least 3 kinds of zinc-containing modules that act as DNA-binding motifs

• All use one or more zinc ions to create a shape to fit an -helix of the motif into the DNA major groove– Zinc fingers– Zinc modules– Modules containing 2 zinc and 6 cysteines

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Homeodomains

• These domains contain about 60 amino acids

• Resemble the helix-turn-helix proteins in structure and function

• Found in a variety of activators

• Originally identified in homeobox proteins regulating fruit fly development

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bZIP and bHLH Motifs

• A number of transcription factors have a highly basic DNA-binding motif linked to protein dimerization motifs– Leucine zippers– Helix-loop-helix

• Examples include:– CCAAT/enhancer-binding protein– MyoD protein

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Transcription-Activating Domains

• Most activators have one of these domains

• Some have more than one– Acidic domains such as yeast GAL4 with 11

acidic amino acids out of 49 amino acids in the domain

– Glutamine-rich domains include Sp1 having 2 that are 25% glutamine

– Proline-rich domains such as CTF which has a domain of 84 amino acids, 19 proline

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Summary

• Eukaryotic activators are composed of at least two domains: a DNA-binding domain and a transcription-activating domain

• DNA-binding domains contain motifs such as zinc modules, homeodomains, and bZIP or bHLH motifs

• Transcription activating domains can be acidic, glutamine-rich or proline-rich

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12.2 Structures of the DNA-Binding Motifs of Activators

• DNA-binding domains have well-defined structures

• X-ray crystallographic studies have shown how these structures interact with their DNA targets

• Interaction domains forming dimers, or tetramers, have also been described

• Most classes of DNA-binding proteins can’t bind DNA in monomer form

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Zinc Fingers

• Described by Klug in GTF TFIIIA

• Nine repeats of a 30-residue element:– 2 closely spaced cysteines followed 12 amino

acids later by 2 closely spaced histidines– Coordination of amino acids to the metal

helps form the finger-shaped structure– Rich in zinc, enough for 1 zinc ion per repeat– Specific recognition between the zinc finger

and its DNA target occurs in the major groove

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Arrangement of Three Zinc Fingers in a Curved Shape

The zinc finger is composed of:

– An antiparallel -strand that contains 2 cysteines

– 2 histidines in an -helix– Helix and strand are

coordinated to a zinc ion

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The GAL4 Protein

• The GAL4 protein is a member of the zinc-containing family of DNA-binding proteins

• Each GAL4 monomer contains a DNA-binding motif with:– 6 cysteines that coordinate 2 zinc ions in a

bimetal thiolate cluster– Short -helix that protrudes into the DNA major

groove is the recognition module– Dimerization motif with an -helix that forms a

parallel coiled coil as it interacts with the -helix on another GAL4 monomer

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The Nuclear Receptors

• A third class of zinc module is the nuclear receptor

• This type of protein interacts with a variety of endocrine-signaling molecules

• Protein plus endocrine molecule forms a complex that functions as an activator by binding to hormone response elements and stimulating transcription of associated genes

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Type I Nuclear Receptors

• These receptors reside in the cytoplasm bound to another protein

• When receptors bind to their hormone ligands:– Release their cytoplasmic protein partners– Move to nucleus– Bind to enhancers– Act as activators

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Glucocorticoid Receptors

• DNA-binding domain with 2 zinc-containing modules

• One module has most DNA-binding residues

• Other module has the surface for protein-protein interaction to form dimers

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Types II and III Nuclear Receptors

• Type II nuclear receptors stay within the nucleus bound to target DNA sites

• Without ligands the receptors repress gene activity

• When receptors bind ligands, they activate transcription

• Type III receptors are “orphan” whose ligands are not yet identified

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Homeodomain-DNA Complex

• Homeodomains contain DNA-binding motif functioning as helix-turn-helix motifs

• A recognition helix fits into the DNA major groove and makes specific contacts there

• N-terminal arm nestles in the adjacent minor groove

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The bZIP and bHLH Domains

• bZIP proteins dimerize through a leucine zipper– This puts the adjacent basic regions of each

monomer in position to embrace DNA target like a pair of tongs

• bHLH proteins dimerize through a helix-loop-helix motif– Allows basic parts of each long helix to grasp the

DNA target site

• bHLH and bHLH-ZIP domains bind to DNA in the same way, later have extra dimerization potential due to their leucine zippers

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12.3 Independence of the Domains of Activators

• DNA-binding and transcription-activating domains of activator proteins are independent modules

• Making hybrid proteins with DNA-binding domain of one protein, transcription-activating domain of another

• The hybrid protein still functions as an activator

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12.4 Functions of Activators

• Bacterial core RNA polymerase is incapable of initiating meaningful transcription

• RNA polymerase holoenzyme can catalyze basal level transcription– Often insufficient at weak promoters– Cells have activators to boost basal

transcription to higher level in a process called recruitment

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Eukaryotic Activators

• Eukaryotic activators also recruit RNA polymerase to promoters

• Stimulate binding of general transcription factors and RNA polymerase to a promoter

• 2 hypotheses for recruitment:– General TF cause a stepwise build-up of

preinitiation complex– General TF and other proteins are already

bound to polymerase in a complex called RNA polymerase holoenzyme

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Models for Recruitment of Preinitiation Complex Components in Yeast

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Recruitment of TFIID

• Acidic transcription-activating domain of the herpes virus transcription factor VP16 binds to TFIID under affinity chromatography conditions

• TFIID is rate-limiting for transcription in some systems

• TFIID is the important target of the VP16 transcription-activating domain

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Recruitment of the Holoenzyme

• Activation in some yeast promoters appears to function by recruitment of holoenzyme

• This is an alternative to the recruitment of individual components of the holoenzyme one at a time

• Some evidence suggests that recruitment of the holoenzyme as a unit is not common

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Recruitment Model of GAL11P-containing Holoenzyme

• Dimerization domain of FAL4 binds to GAL11P in the holoenzyme

• After dimerization, the holoenzyme, along with TFIID, binds to the promoter, activating the gene

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12.5 Interaction Among Activators

• General transcription factors must interact to form the preinitiation complex

• Activators and general transcription factors also interact

• Activators usually interact with one another in activating a gene– Individual factors interact to form a protein

dimer facilitating binding to a single DNA target site

– Specific factors bound to different DNA target sites can collaborate in activating a gene

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Dimerization

• Dimerization is a great advantage to an activator as it increases the affinity between the activator and its DNA target

• Some activators form homodimers but others function as heterodimers

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Action at a Distance

• Bacterial and eukaryotic enhancers stimulate transcription even though located some distance from their promoters

• Four hypotheses attempt to explain the ability of enhancers to act at a distance– Change in topology– Sliding – Looping – Facilitated tracking

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Hypotheses of Enhancer Action

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3C: Method to detect DNA looping

• Chromosome conformation capture (3C) is a technique used to determine if enhancer action requires DNA looping

• Used to test whether two remote DNA regions, such as an enhancer and a promoter, are brought together

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Genomic Imprinting

• Because most eukaryotes are diploid organisms, you would predict that it does not matter which allele of any given gene came form the mother or the father

• This is true in most cases but there are important exceptions

• The differences between the genes resides in how they are modified, or imprinted, differently in females and males

• Evidence exists in mice and humans

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Transcription Factories

• Discrete nuclear sites where transcription of multiple genes occurs

• If two or more genes on the same chromosome are clustered in the same transcription factory, DNA loops would naturally form between them

• Thus, the existence of transcription factories implies the existence of DNA loops in eukaryotic cells

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Evidence for Transcription Factories

• Cook and colleagues counted the number of transcription factories by labeling growing RNA chains in HeLa cells with BrU followed by permeabilization and further labeling with biotin-CTP detected with anti-BrU or anti-biotin primary antibodies followed by secondary antibodies labeled with gold particles

• They concluded that transcription is associated with the clusters, not the single particles

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Complex Enhancers

• Many genes can have more than one activator-binding site permitting them to respond to multiple stimuli

• Each of the activators that bind at these sites must be able to interact with the preinitiation complex assembling at the promoter, likely by looping out any intervening DNA

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Control Region of the Metallothionine Gene

• The metallothionine gene product helps eukaryotes cope with heavy metal poisoning

• Turned on by several different agents• Complex enhancers enable a gene to respond

differently to different combinations of activators• This gives cells exquisitely fine control over their

genes in different tissues, or at different times in a developing organism

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Architectural Transcription Factors

Architectural transcription factors are those transcription factors whose sole or main purpose seems to be to change the shape of a DNA control region so that other proteins can interact successfully to stimulate transcription

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Example of Architectural Transcription Factor: Control region of the human TCR chain gene

• Within 112 bp upstream of the start of transcription are 3 enhancer elements

• These elements bind to:– Ets-1, LEF-1, CREB

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Enhanceosome

• An enhanceosome is a nucleoprotein complex containing a collection of activators bound to an enhancer in such a way that stimulates transcription

• The archetypal enhanceosome involves the IFN enhancer with a structure that involves eight polypeptides bound cooperatively to an essentially straight 55-bp strech of DNA

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DNA Bending Aids Protein Binding

• The activator LEF-1 binds to the minor groove of its DNA target through its HMG domain and induces strong bending of DNA

• LEF-1 does not enhance transcription by itself

• Bending it induces helps other activators bind and interact with activators and general transcription factors

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Insulators

• Insulators can shield genes from activation by enhancers (enhancer blocking activity)

• Insulators can shield genes from repression by silencers (barrier activity)

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Mechanism of Insulator Activity

• Sliding model– Activator bound to an

enhancer and stimulator slides along DNA from enhancer to promoter

• Looping model– Two insulators flank an

enhancer, when bound they interact with each other isolating enhancer

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Model of Multiple Insulator Action

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Summary

• Some insulators have both enhancer-blocking and

barrier activities, but some have only one or the

other

• Insulators may do their job by working in pairs that

bind proteins that can interact to form DNA loops

that would isolate enhancers and silencers so they

can no longer stimulate or repress promoters

• Insulators may establish boundaries between DNA

regions in a chromosome

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12.6 Regulation of Transcription Factors

• Phosphorylation of activators can allow them to interact with coactivators that in turn stimulate transcription

• Ubiquitylation of transcription factors can mark them for – Destruction by proteolysis

– Stimulation of activity

• Sumoylation is the attachment of the polypeptide SUMO which can target for incorporation into compartments of the nucleus

• Methylation and acetylation can modulate activity

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Phosphorylation and Activation: A Model for activation of a CRE-linked gene

Replace this area with Figure 12.33:

A model for activation of a CRE-linked gene

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Model for the Activation of a Nuclear Receptor-Activated Gene

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Ubiquitylation

• Ubiquitylation, especially monoubiquitylation, of some activators can have an activating effect

• Polyubiquitylation marks these same proteins for destruction

• Proteins from the 19S regulatory particle of the proteasome can stimulate transcription

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Activator Sumoylation

• Sumoylation is the addition of one or more copies of the 101-amino acid polypeptide SUMO (Small Ubiquitin-Related Modifier) to lysine residues on a protein

• Process is similar to ubiquitylation

• Results quite different – sumoylated activators are targeted to a specific nuclear compartment that keeps them stable

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Activator Acetylation

• Nonhistone activators and repressors can be acetylated by HATs

• HAT is the enzyme histone acetyltransferase which can act on nonhistone activators and repressors

• Such acetylation can have either positive or negative effects

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Signal Transduction Pathways

• Signal transduction pathways begin with a signaling molecule interacting with a receptor on the cell surface

• This interaction sends the signal into the cell and frequently leads to altered gene expression

• Many signal transduction pathways rely on protein phosphorylation to pass the signal from one protein to another

• This leads to signal amplification at each step

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Three pathways that use CBP/p300 to mediate transcription activation

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Ras and Raf Signal Transduction