RR6-8 (this one is good)

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Questions and Answers

What is the role of the Mediator in eukaryotic transcription?

It recruits RNA polymerase I only.

It degrades RNA after transcription.

It inhibits transcription initiation.

It enhances the interaction of key players. (correct)

What does increased transcription indicate when using in vivo techniques?

Only degraded RNA is present.

Decreased RNA binding to reporter proteins.

No transcriptional activity in the cell.

Active transcription of the gene of interest. (correct)

How does transcription typically occur according to the provided information?

In bursts separated by inactivity. (correct)

Only during the cell cycle phases.

Only from non-enhanced genes.

Continuously without interruption.

What is the significance of the 5’ region sequence during transcription?

It is recognized by a protein fused to a reporter protein. Signup and view all the answers

What does the initial experiment involving the SNAIL gene in Drosophila demonstrate?

Strong enhancers lead to increased initiation events. Signup and view all the answers

What role does the 5’ methylguanosine cap play in mRNA processing?

It protects the mRNA from degradation. Signup and view all the answers

Which components associate with the phosphorylated CTD of RNA Pol II during transcription?

Splicing and polyadenylation factors. Signup and view all the answers

What happens to introns during mRNA processing?

They are spliced out of the mature mRNA. Signup and view all the answers

Which of the following statements about the spliceosome is correct?

It transiently associates with mRNA to facilitate splicing. Signup and view all the answers

During the splicing cycle, which snRNAs interact with mRNA first?

U1 and U2. Signup and view all the answers

What is the outcome of the first transesterification reaction in splicing?

Creation of a lariat structure. Signup and view all the answers

Why are hnRNPs significant in mRNA processing?

They assist in the splicing and transport of mRNA. Signup and view all the answers

What is a key function of the spliceosome's snRNAs?

They recognize and bind to splice sites in the pre-mRNA. Signup and view all the answers

What is the relationship between transcription bursts and P-granules?

Transcription bursts correlate with the formation and dissolution of P-granules. Signup and view all the answers

How does hypoacetylation affect chromatin structure?

It induces chromatin compaction and transcriptional silencing. Signup and view all the answers

What role do histone acetyl-transferases (HATs) play in transcription?

They neutralize electrostatic interactions, allowing transcriptional activation. Signup and view all the answers

Which epigenetic writers modify the histone tails to influence transcription?

Transcriptional activators. Signup and view all the answers

What characterizes heterochromatin?

It is condensed and transcriptionally inactive. Signup and view all the answers

What is the primary function of 5’ capping in mRNA processing?

To provide stability and protection of mRNA. Signup and view all the answers

What mechanism allows pioneer transcription factors to activate genes?

They recruit enzymes that alter the histone configuration. Signup and view all the answers

How do chromatin states influence gene expression?

Through modifications on the histone tails. Signup and view all the answers

What does the term 'epigenetic trait' refer to?

A heritable phenotype without changes to the DNA sequence. Signup and view all the answers

Which of the following conditions is likely to involve transcriptional repression?

Hypoacetylation of histones. Signup and view all the answers

What is the primary role of RAP1 in the context of DNA silencing?

To bind to DNA in the silencer regions and repetitive sequences. Signup and view all the answers

Which proteins interact specifically with hypoacetylated histone tails during the silencing processes?

SIR2, SIR3, and SIR4 Signup and view all the answers

How does the cooperation between RAP1 and SIR1 contribute to DNA silencing?

They bind to silencer regions at silent mating type loci. Signup and view all the answers

What is a key consequence of the binding and activity of silencing proteins on chromatin structure?

Compaction of chromatin, leading to transcriptional silencing. Signup and view all the answers

Which of the following is NOT a function of the SIR proteins in DNA silencing?

Recruiting factors to initiate transcription. Signup and view all the answers

Which step in the splicing cycle is characterized as 'active'?

U1 and U4 exit Signup and view all the answers

What is the immediate outcome of transesterification reactions during splicing?

Joining of exons and removal of introns Signup and view all the answers

Which of the following components is NOT involved in the splicing cycle?

Heterochromatin Signup and view all the answers

What happens to an intron after mature mRNA is released?

It is enzymatically degraded. Signup and view all the answers

What structure is formed when the hydroxyl group at the branch point attacks the 5' phosphate at the first intron residue during splicing?

A lariat structure Signup and view all the answers

Which small nuclear RNA (snRNA) is responsible for contacting the intron splice site border?

U1 Signup and view all the answers

What is the second transesterification reaction that occurs during splicing?

The 3' end of the exon attacks the 5' end of the next exon Signup and view all the answers

Which snRNA is involved in forming the active spliceosome complex along with U2 and U6?

U5 Signup and view all the answers

What motif is essential for U2 snRNA's role in splicing?

The branch point region Signup and view all the answers

What is the main role of the MAT locus in yeast mating?

It determines the overall mating type of the yeast. Signup and view all the answers

Which loci are transcriptionally silent copies of the α mating type?

HML and HMR Signup and view all the answers

What process is described by the transfer of material from HMR to the MAT locus?

Non-reciprocal recombination Signup and view all the answers

Where are the HML and HMR loci located within the yeast chromosome structure?

Near the left and right telomeres Signup and view all the answers

Which statement accurately describes the role of HML and HMR loci in the mating type system of yeast?

They provide a source of silent mating type information for MAT. Signup and view all the answers

What is the initial step in the CHIP technique used to bind proteins to chromatin?

Crosslinking Signup and view all the answers

Which process is utilized at the final stage of the CHIP technique to analyze the released DNA fragments?

Next-Generation Sequencing Signup and view all the answers

In CHIP, what is the purpose of using an antibody during the process?

To bind the specific protein of interest Signup and view all the answers

Which application of CHIP allows for a comprehensive understanding of the entire genome?

Genome-Wide Analysis Signup and view all the answers

What happens to the DNA after it is immunoprecipitated in the CHIP method?

It is released from the protein-antibody complex Signup and view all the answers

What role does Serine 2 phosphorylation play in the context of RNA Pol II transcription?

It recruits specific splicing and polyadenylation factors. Signup and view all the answers

Which factors are specifically associated with the phosphorylated C-terminal domain (pCTD) of RNA Pol II?

Splicing and polyadenylation factors Signup and view all the answers

How does the length of the CTD influence its function during transcription?

It enables multiple proteins to bind simultaneously. Signup and view all the answers

Which statement correctly describes the process of capping in relation to RNA Pol II transcripts?

Capping enzymes bind to the pCTD during transcription. Signup and view all the answers

What is the primary function of the proteins illustrated in the CTD phosphorylation process?

They modify the CTD, influencing transcription stages. Signup and view all the answers

What length of nascent mRNA is required for the addition of the 5' cap?

25 nucleotides Signup and view all the answers

What enzyme is responsible for adding the 5' methylguanylate cap to mRNA?

Capping enzyme dimer Signup and view all the answers

What specific structural feature distinguishes the 5' methylguanylate cap?

7-methylguanosine base Signup and view all the answers

Which factor is exchanged during the process of elongation in relation to capping?

NELF for PAF Signup and view all the answers

Which of the following functions is NOT associated with the 5' cap on mRNA?

Initiation of transcription Signup and view all the answers

What effect does activator-directed histone hyperacetylation have on gene expression?

It promotes the formation of transcription complexes on DNA. Signup and view all the answers

Which proteins are primarily associated with the repressor-directed histone deacetylation process?

Rpd3 Signup and view all the answers

What is the primary consequence of histone deacetylation on DNA structure?

Tightening of DNA around histones, suppressing gene expression. Signup and view all the answers

Which of the following statements accurately describes the action of histone acetyl-transferases (HATs)?

They catalyze the transfer of acetyl groups to histone N-terminal tails. Signup and view all the answers

Study Notes

Mechanisms of Transcriptional Activation, Epigenetics & RNA Processing

The lecture covers mechanisms of transcriptional activation, epigenetics, and RNA processing.
Midterm viewing sessions are scheduled for November 9th and 13th, from 6:30-8:30. Student ID is required.
Scantrons are not available for the midterm.
Fill out a survey on myCourses regarding midterm grading concerns or questions. The survey is available until November 17th.
Tutorials cover material from the week of the lecture.

Mechanisms of Transcriptional Activation and Initiation (RR6)

The Mediator enhances the interaction of key players in eukaryotic transcription.
It is made up of multiple subunits that interact with activation domains.
Mediator flexibility links cis-acting elements to RNA Pol II.
Chromatin loops are induced by the Mediator.
The Mediator facilitates the formation of the pre-initiation complex (PIC).

What Increased Transcription Looks Like

An in vivo technique examines actively transcribed RNA (not degraded RNA).
A protein fused to a reporter protein is added to the 5' region of the target gene.
Transcription of the target gene results in the protein binding the region fused to a reporter protein, which is visible.

Transcription Occurs in Bursts!

Transcriptional initiation from highly transcribed genes happens in bursts with periods of no transcription between them.
An experiment using the SNAIL gene in Drosophila, with a strong enhancer, showed bursts of transcription.

Expression of Reporter Increased and Decreased Over Time

The expression/activity of a reporter protein shows increased and decreased activity over time, showing bursts of activity.
This suggests a Burst Hypothesis rather than a Flux Hypothesis.
The burst frequency correlates with transcription efficiency (enhancer strength).

P-granules

Proteins involved in transcription frequently colocalize in puncta (clusters) due to liquid-liquid condensation.
P-granules form through liquid-liquid condensates, involving high concentrations of macromolecules (DNA, RNA, proteins, and other molecules), influenced by factors like concentration, electrostatic interactions, post-translational modifications, and intrinsically disordered proteins, which have a transient expression.

Dynamic Kissing Model

Enhancers interact with promoters along the surface of transcriptional condensates.
Bursts of transcriptional activation may correlate with the formation and dissolution of P-granules.

Chromatin, Epigenetics, and the Histone Code (RR7)

DNA is packaged with proteins (same mass) to form chromatin.
DNA associates with histones, creating nucleosomes with protruding tails.
Heterochromatin is condensed, inactive chromatin near centromeres and telomeres, and less accessible to transcriptional machinery.
Euchromatin is less condensed, accessible chromatin, allowing for transcriptional machinery access.

Silent Mating Type Loci

Yeast mating type is controlled by three loci on chromosome III.
The MAT locus is the active, transcribed mating type.
The HML/HMR loci are silent copies of the a or α mating type near telomeres.
Non-reciprocal recombination transfers the HML or HMR mating type to the MAT locus.

Silencing Proteins Make DNA Inaccessible

RAP1 binds DNA in silencer regions and telomeres.
SIR1 (Silent Information Regulator), works with RAP1, aiding in binding to the silencer region during silent mating.
SIR2, SIR3, and SIR4 bind to hypoacetylated histone tails (H3/H4) and form complexes with telomeric DNA, inducing chromatin compaction and silencing; this is done through nucleosome condensation, which involves multiple telomeres associating.

Epigenetic Marks

Modifications on H3/H4 tails are linked to chromatin states and are often stably heritable.
Epigenetic traits change the phenotype without altering DNA sequence.
Epigenetic writers introduce chemical modifications on DNA/histones.
Epigenetic readers interpret these modifications.

CHIP against Histones

Reversible crosslinking agents isolate chromatin-bound proteins and DNA for analysis.
Antibodies identify specific proteins, determining the DNA sequence bound to them via known primers for focused analysis of affected regions, and specific genes; NGS (Next-Generation Sequencing) permits analysis of the entire genome.

Histone (De)Acetylation

Transcriptional activators recruit histone acetyl-transferases (HATs) that neutralize the electrostatic forces between histone N-terminals and the DNA backbone, facilitating complex formation for transcription.
Transcriptional repressors recruit histone deacetylases (HDACs) to deacetylate histones, making the complex less accessible for transcription.

Pioneer Transcription Factors

DNA-binding transcriptional activators interact with exposed sequences on the histone octamer.
The binding energy drives DNA unwrapping from nucleosomes to make DNA accessible from the nucleosomes.
They recruit enzymes to alter neighboring histone configuration.
Necessary for gene activation in highly condensed chromatin.

RNA Processing Pt. 1 (RR8)

mRNA processing involves modifications at the 5' and 3' ends (to ensure stability and protection) of pre-mRNA.
Three major co-transcriptional steps:
- 5' Capping
- 3' Cleavage and Polyadenylation
- RNA splicing.

mRNA Capping

The nascent mRNA emerges from the RNA exit channel and reaches a length of 25 nucleotides.
A 5' methylguanalate cap is added by a capping enzyme.
The capping enzyme interacts with the CTD domain of RNA polymerase II for coordination of the process.
The cap protects the mRNA, facilitates nuclear export, and allows mRNA recognition by translation factors.

CTD Phosphorylation

The length of the CTD allows for multiple proteins to associate with RNA polymerase II simultaneously. These proteins involved in capping, splicing, and polyadenylation are facilitated by the length of the CTD.
Enzymes needed to add the 5' cap, and to regulate spliceosome assembly and activity associate with the CTD.
Splicing and polyadenylation factors associate with the CTD.

mRNA Splicing

hnRNPs (heterogeneous ribonucleoprotein particles) are nuclear proteins that associate with mRNA from RNA polymerase II.
They associate with RNAs via RNA-binding domains.
The proteins regulate pre-mRNA splicing, transport mRNAs from the nucleus, etc.
Introns are removed from mature mRNAs to prevent errors and unwanted translation from non-coding regions; they are often not found in mature mRNAs.
Splicing of mRNAs removes introns and joins exons to produce the mature mRNA product.
Various snRNAs (snRNPs) are transient components of the spliceosome which transiently associate with each other and splice sites.

mRNA Splicing (Spliceosome)

The spliceosome includes transiently interacting snRNAs (U1, U2, U4, U5,U6).
U1 contacts the intron splice site, and U2 contacts the branch point region, enabling spliceosome assembly, leading to splicing.
Two transesterification reactions occur for removing introns and joining exons to form a mature mRNA. The OH group of branch point attacks the 5' phosphate and the 3' end of exon attacks the 5' end of the following exon, leading to lariat formation and intron removal.

Splicing Cycle

U1 and U2 interact with mRNA, initiating spliceosome assembly.
U4, U5, and U6 are recruited and U1 and U4 are released in the active form of the spliceosome.
Transesterification reactions join exons and remove the intron, via enzymatic reactions.
Spliceosome is released and the intron is degraded.

Practice Questions

Changes in elongation factors (NELF) are dependent on a balanced interaction/regulation with other functional counterparts (e.g., DSIF, PAF, etc.)
Factors influencing liquid-liquid condensates (e.g., protein concentration, macromolecule interactions, electrostatic interactions, post-translational modifications, intrinsically disordered proteins, transient expressions, etc.) influence the formation of P-granules, which are thought to be involved in transcription.
Cleavage and polyadenylation occur before splicing, for short transcription units.
Introns are removed from mature mRNAs to prevent errors and unwanted translation from non-coding regions.
The Flux and Burst Transcription hypotheses were discussed and the burst hypothesis favoured in some cases to account for burst-like transcription of genes.

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Test your understanding of the mechanisms of transcriptional activation, epigenetics, and RNA processing in this quiz. Explore key concepts such as the role of the Mediator and the formation of the pre-initiation complex. Ideal for students preparing for the midterm examination.