Chapter 21 - The Transcriptional Regulation of Gene Expression in Eukaryotes

What is a key difference in regulation between eukaryotes and prokaryotes?

Regulation occurs through all the steps seen in prokaryotes and more, including mRNA splicing.

What is the most regulated step in eukaryotic gene expression?

The initiation of transcription.

What role do histones play in eukaryotic gene regulation?

Histones are involved in the formation of nucleosomes, which can be rearranged or modified.

How does chromatin structure affect DNA accessibility?

DNA can be concealed or accessible depending on chromatin structure.

What is the relationship between regulatory sequences and eukaryotic genes?

Regulatory sequences are in higher numbers and further from the genes, containing the regulator binding sites.

What is the state of eukaryotic genes in the absence of regulatory proteins?

Eukaryotic genes are generally inactive.

What are the three mechanisms that control chromatin structure?

1) ATP-dependent changes in nucleosome positioning, 2) Posttranslational chemical modifications of histones, 3) Substitution of specialized histone variants.

What effect does acetylation by histone acetyltransferases (HATs) have on chromatin?

It leads to the decondensing of chromatin.

What effect does methylation have on chromatin structure?

It causes chromatin to become tightly closed.

What are specialized histone variants?

Proteins homologous to common histones, with extensions that have different functions.

What are histone modifications?

Modifications to the C-terminal tail of histones in active chromatin and heterochromatin.

What is the effect of methylation on histones?

Methylation on specific Lys and Arg residues in the tail inactivates chromatin for transcription.

What is the effect of phosphorylation on histones?

Phosphorylation occurs on specific Ser and Thr residues.

What is the effect of acetylation on histones?

Acetylation on specific Lys and Arg residues in the tail activates chromatin for transcription.

What is ubiquitination or sumoylation in relation to histones?

Ubiquitination or sumoylation occurs on specific Lys residues.

What is heterochromatin?

About 10% of the chromatin in a cell that is more condensed and transcriptionally inactive.

What is euchromatin?

Less condensed chromatin where genes can be transcriptionally active.

How can euchromatin be identified?

Euchromatin can be identified by nucleases such as DNase I.

What are hypersensitive sites?

Sequences sensitive to DNase I where DNA is not packaged in its normal nucleosomal structure.

What do hypersensitive sites correspond to?

They correspond to binding sites for regulatory proteins.

What do eukaryotes have an increased number of?

Regulatory proteins

What do regulatory proteins bind to?

Regulator binding sites

What is true about the number of regulatory sites in higher order organisms?

They have a higher number of regulatory sites, not longer sites

Where can regulatory sequences be found in relation to the promoter?

Upstream and downstream from the promoter

What separates genes from the promoter and activator?

Insulators or boundary elements

What are enhancers in the context of regulatory sequences?

Units of regulator binding sites

What is required for RNA polymerase initiation in eukaryotes?

Activators

What problem do eukaryotes face regarding activator recognition?

Sequences recognized by activators may occur at random sites

How do eukaryotes overcome the problem of random activator sites?

By having multiple regulatory sites

What are upstream activator sequences (UAS) in yeast?

Enhancers where activators bind to promoters

What increases with the complexity of an organism?

Promoter complexity

What is required for the binding of RNA polymerase II?

General transcription factors (GTF)

What do DNA binding transcription activators bind to?

Enhancers or UAS to facilitate transcription

What role do coactivators play in transcription?

They act as a scaffold between other proteins

Can proteins act as activators at one promoter and repressors at another?

Yes

How do activators typically interact with RNA polymerase?

They do not often directly interact

What can activators recruit to stimulate RNA polymerase and elongation factors?

Protein complexes like the pTEFb complex

What must RNA Pol II do to initiate transcription?

RNA Pol II must be recruited to the promoter and form the PIC.

What is the role of the TATA box in transcription initiation?

Typically starts the transcription process with the binding of the TBP.

What is TBP and its role in transcription?

TBP (TATA-binding protein) is a general transcription factor that is part of TFIID, which recruits more GTFs.

What is required for a functional PIC?

The help of activators and coactivators is required for a functional PIC.

What are examples of coactivators in transcription?

Coactivators can include TFIID and the Mediator complex.

How does DNA looping assist in transcription?

DNA looping assists with the interaction of proteins that are far away.

What proteins promote DNA looping?

High-mobility group (HMG) proteins promote DNA looping.

What is the role of the Mediator in transcription?

Mediator is needed for transcription activator and repressor proteins to work.

How does Mediator function in relation to RNA Pol II?

Mediator serves as a bridge between the general RNA Pol transcription machinery and gene-specific activators.

What does Mediator help stimulate in transcription?

Mediator helps stimulate the phosphorylation of the CTD by TFIIH.

Where do activator interactions occur in the Mediator complex?

Activator interactions occur mainly in the tail region.

Where do RNA Pol interactions occur in the Mediator complex?

RNA Pol interactions occur mainly at the head and middle regions.

What is the role of CDK8/Srb8-11 in transcription?

CDK8/Srb8-11 is a negative regulator and must be missing for Mediator to interact with RNA Pol II.

What is required for transcription initiation and elongation in eukaryotes?

Eukaryotes require multiple proteins for transcription initiation and elongation.

What can cause RNA Pol to pause downstream from the start site?

Some sequences can pause RNA Pol downstream from the start site, requiring factors to continue elongation.

What is the role of HSF in the activation of the HSP70 gene in Drosophila?

HSF is a second activator required to continue with elongation; otherwise, transcription stalls.

How does HSF facilitate the continuation of transcription elongation?

HSF recruits P-TEFb to phosphorylate the CTD of the RNA Pol tail.

How are GAL genes organized in yeast?

Genes for metabolism are spread through multiple chromosomes in yeast.

Do yeast have operons like bacteria?

No, yeast do not have operons like in bacteria.

What is the role of Gal4p in yeast?

Gal4p is a transcription activator that controls gene expression.

What is the function of Gal80p?

Gal80p is an inhibitor that regulates gene expression.

What is the role of Gal3p in yeast?

Gal3p acts as a ligand sensor in the regulation of GAL genes.

What do Gal4p dimers bind to?

Gal4p dimers bind to a 17mer called upstream of GAL1.

What are the domains of Gal4p from N to C?

Zn2Cys6 binuclear cluster DBD, dimerization region, activating region (AD) I, activating region (AD) II.

What is included in the activating region (AD) II of Gal4p?

The Gal80 binding site is included in the activating region (AD) II.

Which proteins assist in activating GAL1?

Proteins such as Mig1, Tup1, and Gal80 assist in activating GAL1.

What is the function of Mig1 in yeast?

Mig1 is a multicopy inhibitor of Gal1 and acts as a global repressor.

What role does Tup1 play in the regulation of GAL genes?

Tup1 is a corepressor that binds to Mig1.

What prevents GAL1 transcription in the absence of galactose?

Gal80 dimers bind to the Gal80 binding site on Gal4p, preventing activating region II from interacting with components of the transcription machinery.

What happens to GAL1 transcription in the presence of galactose?

Gal3 binds to Gal80 causing Gal80 dimers to dissociate and be released from Gal4p, allowing transcription of GAL1.

How does glucose affect Mig1 regulation of GAL1 transcription?

In the absence of glucose, Mig1 is phosphorylated and stays in the cytoplasm, allowing transcription. In the presence of glucose, Mig1 is no longer phosphorylated and can enter the nucleus to inhibit transcription.

What condition induces GAL1 transcription?

Presence of galactose and absence of glucose.

What is the function of the Gal4p transcription activator?

It stimulates transcription from a reporter gene with a UASGAL site upstream from the transcription initiation site.

Does the Gal4p fragment from residues 1-100 stimulate transcription of the reporter gene?

No, it does not stimulate transcription.

Does the Gal4p fragment from residues 100-881 stimulate transcription of the reporter gene?

No, it does not stimulate transcription.

What is the composition of the LexA-Gal4p hybrid protein?

It consists of the DNA binding domain (DBD) from LexA and the activation domain (AD) from Gal4p.

Does the LexA-Gal4p hybrid protein stimulate transcription from a reporter gene?

Yes, it stimulates transcription from a reporter gene with a LexA site upstream from the transcription initiation site.

What controls the expression of a-specific genes in yeast?

a1 and Mcm1

What controls the expression of α-specific genes in yeast?

α1, α2, and Mcm1

What controls the expression of specific genes in a/α cells?

a1, α2, and Mcm1

Where are the regulators for mating type in yeast expressed?

MAT locus

What mechanism is used for movement in controlling mating type in yeast?

Site-specific recombination (SSR)

What can eukaryotic transcription factors form to increase possibilities?

Homodimers and heterodimers

What is the AP-1 transcription activators family composed of?

Fos, Jun, and ATF

What type of dimerization do AP-1 transcription activators use?

Basic leucine zipper type

What influences the regulation by AP-1 transcription activators?

Many external stimuli

What are insulators in genetics?

Insulators are DNA sequences that control the action of activators.

What is the function of insulators in relation to enhancers and promoters?

Insulators are placed between an enhancer and promoter to inhibit activation of that gene.

What do insulators and CTCF proteins do?

They block communication between the enhancer and promoter without repressing the promoter or inhibiting the activator's activity.

How do insulators affect transcriptional silencing?

Insulators may inhibit the spreading of transcriptional silencing due to chromatin modifications.

What is an enhanceosome?

An enhanceosome is a stable, tightly folded nucleoprotein formed by cooperating activators.

When is the INFβ gene activated?

The INFβ gene is activated upon viral infection.

Which activators are needed for INFβ gene activation?

NF-κB, IRF, and Jun/ATF are needed for INFβ gene activation.

What role does the architectural protein HMGA1 play in enhanceosome assembly?

HMGA1 binds to the enhancer and helps enhanceosome assembly by straightening the DNA.

What is the role of the co-activator CBP-p300 in enhanceosome function?

CBP-p300 recruits nucleosome-remodelers and the transcriptional machinery.

What do transcriptional repressors not normally bind to?

Site overlapping RNA polymerase binding site

How can a repressor prevent an activator from binding?

Repressor binds to its site and does not allow the activator to bind to its site

What happens when a repressor and activator physically interact?

The activator loses its activating region

How can a repressor interact with the transcriptional machinery?

Repressor binds and interacts with the transcriptional machinery

What is the most common way a repressor functions?

Recruitment of histone modifiers

What do transcriptional repressors do?

They recruit nucleosome modifiers that compact chromatin or remove groups that the transcriptional machinery can identify.

What is the role of histone deacetylase (HDAC) in transcription?

HDAC removes acetyl from histone tails, preventing transcription from cryptic promoters.

What effect does the addition of a methyl group to the histone tail have?

It contributes to changes in DNA and histone that lead to silencing.