Class 3 transcript DS

Core Concepts & Review

Transcription Review

A three-step process:

1) Initiation: Assembly of general transcription factors (GTFs) and RNA Pol II at the promoter (e.g., via TBP binding to the TATA box). TFIIH helicase activity opens the DNA.

2) Elongation: Phosphorylation of Ser5 on Pol II's CTD triggers promoter clearance. After initial pausing, phosphorylation of Ser2 promotes processive transcription.

3) Termination: Triggered by sequence signals (e.g., AAUAAA) recruiting cleavage factors or via the "torpedo model" where an exonuclease degrades uncapped RNA and dislodges Pol II.

Enhancer Question

Enhancers bind to the initiation complex through mediator and other cofactors.

Their binding is facilitated by sequence-specific transcription factors and is a key focus of today's lecture on regulation.

Purpose of Transcriptional Pausing

The initial pausing after synthesizing 30-50 nucleotides is a regulatory checkpoint.

It ensures all necessary factors are present and properly assembled before committing to full elongation, allowing for precise control of gene expression.

Operon vs. Enhancer

An operon is a prokaryotic genetic unit where multiple co-regulated genes are transcribed together into a single mRNA molecule.

An enhancer is a eukaryotic cis-regulatory DNA sequence that binds transcription factors to increase transcription of a specific gene from a distance, often through DNA looping. They are not analogous; operons are about gene organization, while enhancers are about regulatory control.

Transcription Factors: Types and Functions

Transcription Factors (TFs)

Proteins that control gene expression by binding to specific DNA sequences to regulate the rate of transcription. They are the key players in determining when and where a gene is turned on.

General Transcription Factors (GTFs)

A set of proteins (e.g., TFIID, TFIIB, TFIIH) required for the initiation of transcription at all RNA Polymerase II promoters.

They assemble the pre-initiation complex (PIC) at the core promoter.

Sequence-Specific Transcription Factors

Proteins that bind to specific DNA sequences, primarily in enhancer regions, to modulate the transcription of specific genes.

They are responsible for the precise spatial and temporal control of gene expression in response to developmental cues, environmental signals, or cell type.

DNA Binding Domain

The region of a transcription factor protein that directly contacts and binds to a specific DNA sequence motif (e.g., in an enhancer).

Activation Domain

The region of a transcription factor protein that recruits other components of the transcription machinery, such as GTFs, Pol II, or cofactors (e.g., Mediator), to increase the rate of transcription initiation.

Transcriptional Cofactors

Proteins that do not bind DNA directly but facilitate transcription through protein-protein interactions.

They are recruited by sequence-specific transcription factors to mediate interactions with the general transcription machinery.

Example: Mediator.

Mediator

A large multi-subunit cofactor complex (20-26 proteins) that acts as a molecular bridge.

It connects sequence-specific transcription factors bound at enhancers with RNA Pol II and the GTFs at the promoter.

It also stimulates the phosphorylation of Ser5 on Pol II's CTD, which is crucial for initiation.

Enhancers: Properties and Mechanisms

Enhancers

DNA sequences, often located far from the gene they regulate (upstream, downstream, or in introns),

that contain clusters of binding sites for sequence-specific transcription factors.

They dramatically increase transcription from a promoter.

Key Properties of Enhancers

1. Can be hundreds of base pairs long and

2. contain multiple binding sites.

3. Often bind transcription factors with degenerate (imperfect) sequence specificity, allowing for nuanced regulation.

4. Are modular and autonomous; they can function independently.

5. Can work in combination with other enhancers in specific times and spaces, which is crucial for the complexity of multicellular organisms.

6. Their activity is heavily influenced by chromatin structure (nucleosomes).

Key Properties of Enhancers 1

1. Can be hundreds of base pairs long and

2. contain multiple binding sites.

Key Properties of Enhancers 2

1. Often bind transcription factors with degenerate (imperfect) sequence specificity, allowing for nuanced regulation.

Key Properties of Enhancers 3

1. Are modular and autonomous; they can function independently.

1. Can work in combination with other enhancers in specific times and spaces, which is crucial for the complexity of multicellular organisms.

Key Properties of Enhancers 4

1. Their activity is heavily influenced by chromatin structure (nucleosomes).

Combinatorial Control

The concept that a single gene's expression is controlled by the combined effects of multiple transcription factors binding to its enhancer(s). The specific mix of factors present in a cell determines the gene's output, allowing for immense diversity from a limited set of regulators.

Positive Regulation

When the binding of transcription factors to an enhancer increases the rate of transcription. Mechanisms include:

Cooperative Binding: Binding of one TF facilitates the binding of a second, neighboring TF.

Heterodimer Formation: Two different TFs bind to each other first, forming a complex that then has higher affinity for the enhancer.

Co-binding: An intermediary cofactor facilitates the simultaneous binding of multiple TFs.

Co-occupancy: Rapid, alternating binding of different TFs to the same site to finely tune the rate of transcription.

Cooperative Binding

Binding of one TF facilitates the binding of a second, neighboring TF.

Heterodimer Formation

Two different TFs bind to each other first, forming a complex that then has higher affinity for the enhancer.

Co-binding

An intermediary cofactor facilitates the simultaneous binding of multiple TFs.

Co-occupancy

Rapid, alternating binding of different TFs to the same site to finely tune the rate of transcription.

Negative Regulation

When the binding of transcription factors to an enhancer decreases the rate of transcription (repression). This can occur by:

Direct Repression: A repressor TF prevents the assembly or function of the PIC.

Weaker Activators: The binding of a less effective activator TF reduces the transcription rate compared to a stronger activator. This is common in dynamic processes like immune response, where genes must be rapidly turned on and then off.

Chromatin & Nucleosomes

The packaging of DNA into nucleosomes (DNA wrapped around histone proteins) presents a fundamental barrier to transcription factor binding. Enhancer activity is therefore intrinsically linked to chromatin accessibility.

Pioneer Transcription Factors

A special class of transcription factors that can bind to their target sequences even on nucleosome-bound DNA. Their binding can displace or reposition nucleosomes, making the DNA accessible for other transcription factors to bind.

Nucleosome-Blocking Transcription Factors

Transcription factors whose binding prevents other factors from accessing the DNA, effectively acting as repressors by blocking enhancer sites.

DNA Bending/Looping

The binding of transcription factors can induce bends in the DNA helix. This facilitates long-range interactions via DNA looping, bringing distant enhancers into close physical proximity with the promoter and initiation complex.

Enhancer Architecture Models

Enhancer Architecture

The concept that the specific arrangement, order, spacing, and composition of transcription factor binding sites within an enhancer determines its function.

Enhancerosome Model

A model of enhancer function where the precise order, orientation, and spacing of transcription factor binding sites is absolutely critical for function. The enhancer acts like a precise lock-and-key mechanism; any perturbation (mutations, spacing changes) disrupts its ability to activate transcription.

Billboard Model

A model of enhancer function where only the composition (the number and type of transcription factor binding sites) matters. The order, orientation, and spacing of the sites are flexible and not critical for function. The enhancer acts like a billboard displaying information; the message is what's important, not the exact layout.

Experimental Analysis: Enhancer Reporters

Enhancer Reporter Assay

A powerful experimental method used to identify and characterize enhancer sequences. A candidate DNA sequence is placed upstream of a core promoter driving a reporter gene (e.g., GFP, luciferase). If the candidate sequence functions as an enhancer, it will increase expression of the reporter gene. This assay tests whether a sequence is sufficient to drive transcription.

Using Reporters to Test Models

The enhancerosome and billboard models can be tested by engineering mutations in an enhancer and measuring the effect on reporter gene expression:

Enhancerosome Prediction: The enhancer will be highly sensitive to mutations that change the order, orientation, or spacing of binding sites. Expression will be lost ("Not Expressed").

Billboard Prediction: The enhancer will be robust to mutations as long as the same types of binding sites are present. Expression will be maintained ("Expressed").

Interpretation of Mutants

Swap/Inversion Mutants (e.g., A<>C swap, full reversal): Likely disrupts an enhancerosome (Not Expressed) but not a billboard (Expressed).

Spacing Mutants (e.g., +20 bp, +1 bp): Likely disrupts an enhancerosome (Not Expressed) but not a billboard (Expressed), though very small changes (+1 bp) might be tolerated.

Deletion Mutants (e.g., remove 2nd A site): Could disrupt both models if the site is essential (Not Expressed), or only disrupt the enhancerosome if the billboard only requires one A site (Enhancerosome: Not Expressed, Billboard: Expressed). The outcome depends on the additive requirement of sites.

Synthesis & Context

Biological Context

The choice between enhancerosome and billboard models is not absolute; it is gene-context and condition-dependent. Some genes require the precise architecture of an enhancerosome for strict regulation, while others use the flexible billboard model for robust expression. This is an area of active research.

From Mechanism to Phenotype

The combination of specific transcription factors, their interactions at enhancers, and the underlying chromatin state integrates diverse signals (developmental, environmental) to precisely control transcription. This sophisticated regulatory layer is what allows a complex genotype to produce a specific phenotype in a multicellular organism.