Structure of the DNA-binding motifs of activators

Chapter 12

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

DNA-binding domains

• 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

• 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 α-helix of the motif into the DNA major groove– Zinc fingers – TFIIIA and Sp1– Zinc modules – Glucocorticoid receptor– Modules containing 2 zinc and 6 cysteines –

GAL4

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

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

Transcription-Activation Domains

• Acidic domains: GAL4

• Glutamine-rich domains: Sp1

• Proline-rich domains: CTF

• Structure and function – not clearly related

The GAL4 Protein

• Yeast activator controls a set of genes responsible for metabolism of galactose

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

Nuclear receptor

• Zinc module - 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

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

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

- Bind ligands - they activate transcription

• Type III receptors - ligands are not yet identified

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

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

Models for Recruitment

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

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 (homework)– Change in topology– Sliding – Looping – Facilitated tracking

Hypotheses of Enhancer Action

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 - by looping out any intervening DNA

Control Region of the Metallothionine Gene

• Gene product helps eukaryotes cope with heavy metal poisoning

• Turned on by several different agents

Architectural Transcription Factors

Architectural transcription factors are those transcription factors - change the shape control region so that other proteins can interact successfully to stimulate transcription

Enhanceosome

• An enhanceosome is a complex of enhancer DNA with activators contacting this DNA

• An example is the HMG that helps to bend DNA so that it may interact with other proteins

Examples of Architectural Transcription Factors

• Besides LEF-1, HMG I(Y) plays a similar role in the human interferon-b control gene

• For the IFN-b enhancer, activation seems to require cooperative binding of several activators, including HMG I(Y) to form an enhanceosome with a specific shape

Homework

• Explain the four hypotheses of the ability of enhancers to act at a distance.

• What are insulators? Explain the functions of insulators. Explain mechanism of insulator activity.

Study material for exam

• Structure of activator• Three types of DNA binding motif• Three types of transcription activation domain• What is enhancer? Two types – architectural factors

and enhanceosome• Recruitment• Insulator• Ubiquitylation• Sumoylation

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

Ubiquitylation

• Ubiquitylation - monoubiquitylation of some activators can have an activating effect

• Polyubiquitylation marks these same proteins for destruction

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

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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