Molecular Bio 330 - Exam 3: Lec 7 -

What is the primary function of a transcription activator in eukaryotic gene regulation?

It is a regulator protein that binds to DNA to turn genes on by increasing transcription.

In eukaryotic DNA, what is an enhancer?

A regulatory DNA sequence that binds activators to stimulate transcription from a distance.

Where can enhancers be located relative to the gene they regulate?

They can be located on either side of the gene, at a distance, or even within the gene itself.

A regulatory DNA sequence that a repressor protein binds to is called a _____.

Silencer

How do transcription regulators bound at distant enhancers reach the promoter vicinity?

Through DNA looping, which is often mediated by the Mediator complex.

In multicellular organisms, what is the main cause of the distinct characteristics found in different cell types?

The differential regulation of genes transcribed by RNA polymerase II.

The concept that gene expression is regulated by the combined action of multiple transcription factors is known as _____.

Combinatorial Control

Why does a liver cell produce albumin while a lens cell produces crystallin despite having identical genomes?

Each cell type contains a unique combination of specific transcription factors (activators) required for those specific genes.

Different combinations of TFs determine?

When, where, and how strongly a gene is expressed

Differential gene expression allows?

Different cells to produce only the proteins they need

In the liver cell, the “liver-specific” activators do what?

Bind to the albumin enhancer, causing the DNA to bend to allow the activators to interact with the promoter and initiate transcription of albumin

In the lens cell, the “lens-specific"“ activators are?

Present and bind to the crystallin enhancer, triggering the expression of crystallin

Cis-acting regulatory elements

DNA sequences located on the same chromosome as the gene they regulate that serve as binding sites for proteins

• act relatively locally

What is required for accurate regulated transcription of genes?

• Promoters

• Enhancers

• Solencers

• Insulators

Trans-acting factors

Proteins encoded elsewhere in the genome that diffuse to bind to specific target cis-acting DNA sequences.

• act at a distance

How can the combination of control elements activate transcription?

Only when the appropriate activator proteins are present

What is the specific function of an insulator DNA sequence?

It blocks or insulates the effect of an enhancer on a promoter when positioned between them.

Where is an insulator located and why?

Between an enhancer and the promoter of a non-target gene to prevent the enhancer from influencing the transcription of the non-target gene

How do insulators help organize gene regulation in the genome?

They serve as boundary elements that help create distinct neighborhoods of gene regulation.

Core promoters vs Proximal-promoter elements?

Core promoter: Determines accurate initiation of transcription

Proximal-promoter elements: Modulate the efficiency of basal levels of transcription

In a reporter gene assay, what is the 'reporter gene' used for?

It is a gene attached to a regulatory sequence (like a promoter or enhancer) of another gene that monitors the timing, location, and strength of gene expression.

If deleting a specific DNA control element causes a significant reduction in reporter gene expression, what was that element's normal role?

To activate transcription (acting as an enhancer or activator binding site).

If deleting a specific DNA control element causes an increase in reporter gene expression, what was that element's normal role?

To suppress or inhibit transcription (acting as a silencer or repressor binding site).

How is transcriptional output (mRNA level) quantified?

• RT-qPCR (measuring mRNA directly), RNA sequencing

• Reporter gene assays

In an experiments based on control elements, what is independent vs dependent variable?

Independent: The possible control element that was deleted/mutated

Dependent: The relative level of reporter gene mRNA

If deletion of a control element causes a reduction in gene expression, what must be the normal role of that control element?

To activate transcription

If the deletion of a control element causes an increase in gene expression, what must be the normal role of that control element?

To suppress or inhibit transcription

What occurs without the control element?

Activators are not able to bind to the enhancer, and the level of gene expression increases

If deleting a region decreases expression, that implies the region had?

If deleting a region increases expression, that implies the region had?

1) Enhancer/activator site

2) Silencer/repressor site

What is required for transcription to start?

TATA box

Purified system vs Nuclear extract?

1) RNA Pol II and the purified general transcription factors

2) Containing most of the proteins from the nuclei of cultured cells

What are the four structural genes and three regulatory genes of the GAL gene system in yeast?

4: GAL 1, 2, 7, 10

3: GAL 3, 4, 80

What happens when there is no galactose vs when galactose is present in the yeast GAL gene family?

No galactose: GAL genes are not transcribed

Galactose present: GAL genes are transcribed

Transcription is activated by GAL4 in response to?

Galactose

What is the function of the GAL80 protein in the absence of galactose?

It acts as a repressor by binding to GAL4 and blocking its activation domain.

In the presence of galactose, what is the function of the GAL 3 protein?

Acts as a signal transducer or galactose sensor and activates GAL4 by inhibiting GAL80, allowing transcription of GAL genes

In the presence of galactose, what is the funciton of the GAL3 protein?

It activates transcription by binding to upstream activating sequences (UAS) of GAL genes to promote their transcription

What positively regulates Gal 1, 2, 7, 10 ?

Gal4 activator

Which Gal blocks, masks, and inhibits Gal4?

Gal80p

How does the GAL3-galactose complex relieve the repression of GAL genes?

It binds to GAL80, causing a conformational change or sequestration that exposes the GAL4 activation domain.

The specific DNA binding site for the GAL4 activator in yeast is called the _____.

Upstream Activating Sequence (UAS_G)

What are the two functional domains found in the GAL4 protein?

A DNA-binding domain (DBD) and an activation domain (AD).

True or False: Attenuation is a common mechanism of gene regulation in eukaryotes.

False (it cannot take place because transcription and translation are spatially separated).

What is the mechanism of galactose binding to Gal3 protein?

This Gal3-galactose complex binds to Gal80 which relieves Gal80 inhibition of Gal4p and then exposes activation domain of Gal4p

What happens if you delete one of the four UAS_G?

Transcription will still occur but it will be weakly/poor

What effect would a deletion of the TATA box (located between $-50$ and $-11$) have on transcription?

It would result in a complete lack of transcription in both crude extracts and purified systems.

Why might a template show high transcription in a nuclear extract but low transcription in a purified system containing only RNA Pol II and general TFs?

The nuclear extract contains specific transcription factors (activators/mediators) that are missing in the purified system.

In promoter analysis, a promoter with a single defined transcription start site and an essential TATA box is categorized as a _____ promoter.

Focused

What would be the effect of a mutation in the $GAL4$ gene that removes the DNA-binding domain?

The GAL4 protein would be unable to bind to the $UAS_{G}$, preventing transcription of $GAL$ structural genes even if galactose is present.

In eukaryotic cells, $mRNA$ level is often quantified directly using which technique?

RT-qPCR or RNA sequencing (RNA-seq).

What is the transcription start site typically designated as in genetic diagrams?

$+1$

In the context of the yeast $GAL$ system, how many $UAS$ binding sites are typically found in the regulatory region?

Four

Which complex is responsible for delivering the effects of multiple transcription regulators to RNA Pol II?

The Mediator complex.

A yeast mutant that continuously expresses $GAL$ genes regardless of galactose presence likely has a mutation in which regulatory gene?

$GAL80$ (loss of the repressor).

How do silencer elements decrease the rate of transcription?

By decreasing the rate of preinitiation complex (PIC) assembly and RNA Pol II release.

What happens to the $GAL4$ protein in the presence of galactose?

Its activation domain is exposed due to the interaction between GAL3, galactose, and GAL80.

In reporter gene experiments, the deleted or mutated control element represents the _____ variable.

Independent

In reporter gene experiments, the relative level of reporter $mRNA$ represents the _____ variable.

Dependent

Eukaryotic transcription initiation is primarily regulated by $trans$-acting factors binding to _____ sites.

$Cis$-acting

What is the typical distance an enhancer can be from a promoter while still modulating transcription?

Up to thousands of base pairs ($bp$).

Which specific protein is required for the transcription of $GAL$ structural genes in yeast?

GAL4 protein

If a promoter region between $-81$ and $-50$ is required for high transcription levels in a crude system, what does this suggest about that region?

It interacts with specific components (activators) found in nuclear extracts but not in purified systems.

How do transcription activators increase the rate of gene expression?

By binding to enhancers or proximal-promoter elements to facilitate the assembly of the transcription machinery.

What is the function of the $5'$ flanking region of a gene?

It typically contains regulatory elements like promoters, enhancers, and silencers that control transcription initiation.

The core promoter determines the _____ of transcription initiation, whereas proximal elements determine the _____.

accuracy; efficiency (or basal level)

What is the consequence of GAL80 masking the activation domain of GAL4?

GAL4 cannot interact with the basal transcription apparatus, preventing transcription.

Which system is used to test if general transcription factors alone are sufficient for high efficiency of a specific promoter?

A purified system (containing RNA Pol II and general TFs only).

Eukaryotic gene regulation is highly influenced by modifications to _____.

Chromatin (including histone modification and DNA methylation).

Which class of $cis$-acting elements is functionally similar to enhancers in yeast?

Upstream Activation Sequences ($UAS$).