LC 6

What is the fundamental difference between eukaryotic DNA and prokaryotic DNA with regard to expression of genes? What is required for appropriate levels of transcription?

Eukaryotic DNA is extensively packed into nucleosomes (chromatin).

By default, eukaryotic genes tend to be repressed.

Positive Regulation (activation)

How is eukaryotic transcription activated?

• DNA-binding activators (analogous to CAP).

• Histone-modifying machinery increases DNA accessibility to transcription machinery.

Where can eukaryotic transcriptional activators bind?

• Proximal promoter: directly upstream of the core promoter.

• Enhancer elements: may be located many kb away from the core promoter.

What are the two general domains of eukaryotic activator proteins?

• DNA-binding domain.

• Activation domain.

• Both domains are within the same polypeptide.

What is the general architecture and functional organization of transcriptional activators?

• DNA-binding domain.

• Domain that interacts with transcriptional machinery.

• Usually function as dimers.

What are the common structural motifs found in DNA-binding domains?

• Helix-turn-Helix

• Zinc finger

• Leucine zipper

How does the helix-turn-helix motif interact with DNA?

• Contacts the DNA-binding site.

• Found in repressor proteins.

What is the structural composition of a zinc finger motif?

• A beta-hairpin followed by an alpha helix.

• Stabilized by a zinc ion.

What is the structure and DNA-binding mechanism of a leucine zipper motif?

• Dimer of extended alpha-helical proteins.

• Proteins bind to each other through hydrophobic interactions.

• Bind to DNA through basic (positively charged) regions.

What are the proposed mechanisms by which eukaryotic transcriptional activation domains stimulate transcription?

1. Physically recruit pol II to the promoter: Activation domains link up directly or indirectly with components of preinitiation complex

2. Stimulate CTD phosphorylation: Activation domains interact with pre-formed preinitiation complex through mediator proteins, stimulating transcription initiation.

3. Recruit enzymes that modify nucleosomes: Making DNA more open/accessible to transcriptional machinery.

What are enhancer elements, and how do they contribute to transcriptional activation?

• DNA regions that may be distant from the core promoter.

• Contain binding sites for transcriptional regulators.

• Multiple activators bind side-by-side and indirectly stimulate preinitiation complex (GTFs/RNA Pol II) assembly.

• The resulting higher-order DNA/protein structure is called an enhanceosome.

What is the Mediator complex, and what is its role in transcription?

• Massive adapter complex composed of numerous distinct polypeptide subunits.

• Communicates between the preinitiation complex (PIC) and other transcriptional regulators.

• Allows activators to indirectly interact with RNA Pol II or PIC components.

How does Mediator contribute to transcriptional activation?

• Interacts with transcriptional activators and the preinitiation complex (PIC).

• Srb8-11 must be displaced for Mediator to have an activating effect.

• Mediator regions include Tail, Middle, Head, and Srb8-11.

What are the two types of nucleosome modification activities recruited by transcriptional activators?

1. Nucleosome remodeling complexes (SWI/SNF)

2. Histone acetyltransferases (HATs)

How do chromatin remodeling complexes (such as SWI/SNF) promote transcription?

• Use ATP energy to physically displace nucleosomes that obstruct transcription machinery.

• SWI/SNF is a 12-subunit (12-polypeptide) complex.

How do histone tail modifications affect chromatin accessibility and transcription?

• Histone tails can be modified with acetyl, methyl, phosphate, or ubiquitin groups.

• Highly acetylated histones → open chromatin → increased transcription accessibility.

• Hypo-acetylated or methylated histones → closed (inaccessible) chromatin.

What enzymes are responsible for adding and removing acetyl and methyl groups on histone tails?

• HATs: Histone acetyltransferases: Add acetyl groups.

• HDACs: Histone deacetylases: Remove acetyl groups.

• MTs: Methyltransferases: Add methyl groups.

• DMs: Demethylases: Remove methyl groups.