Mod 5- Gene Expression In Eukaryotes

Question 1

What is the primary role of transcription factors?

Answer 1

To regulate gene expression by binding to specific DNA sequences.

Question 2

What is the structure of eukaryotic DNA organized into?

Answer 2

Chromatin.

Question 3

What does DNase hypersensitivity indicate regarding nucleosomes?

Answer 3

Nucleosomes are cleared from active chromatin through sliding or eviction.

Question 4

What are the components of histone structure?

Answer 4

N-terminal variable basic arm, non-polar globular domain, C-terminal variable basic arm.

Question 5

What is the significance of post-translational modifications of histones?

Answer 5

Methylation
- CH3 groups added to lys and his
- involved in gene activation and repression, DNA replication

Acetylation
- addition of CH3CO2- group to lysines of core histones (neutralises arms & loosens association with DNA)
- group is negatively charged
- involved in nucleosome assembly and gene activation

Phosphorylation
- addition of PO4- to lysines of H1
- involved in cell division, transcription and chromatin structure

Question 6

Fill in the blank: A class II regulatory region includes a _______.

Answer 6

[TATA box, enhancer (landing pad for proteins), promoter]

Question 7

What are the two domains of a bipartite transcription factor?

Answer 7

Transcription activation domain - interacts with polymerase to activate transcription

DNA binding domain.

Question 8

What types of interactions are involved in transcriptional stimulation in eukaryotes?

Answer 8

Mechanism depends on:

• Long distance interactions facilitated by DNA bending/looping
• Recruitment of general (basal) factors/polymerase
• Recruitment/ co-recruitment of HATs and chromatin modifier complexes.

Question 9

What is the role of signal transduction pathways in transcriptional regulation?

Answer 9

They result in phosphorylation of transcription factors, modulating their activity.

Question 10

True or False: Ligand-receptor interaction at the plasma membrane does not affect transcription factor activity.

Answer 10

False.

Question 11

What are nuclear hormone receptors?

Answer 11

A large superfamily of ligand-dependent transcription factors/receptors.

Activated by hydrophobic molecules

Question 12

What happens when a hydrophobic steroid hormone binds to its receptor?

Answer 12

The receptor translocates to the nucleus to activate transcription.

Question 13

Fill in the blank: _______ is an example of a second messenger in signal transduction pathways.

Answer 13

[cAMP]

Question 14

What is the bipartite nature of sequence-specific transcription factors?

Answer 14

They have a DNA binding domain and a transcription activation domain.

Question 15

What must be modified for transcription factor access?

Answer 15

Chromatin structure.

Question 16

What types of interactions are crucial for gene regulation?

Answer 16

DNA-protein interactions, protein-protein interactions, co-activators/co-repressors.

Question 17

What are some examples of ligands for nuclear hormone receptors?

Answer 17

• Estrogens
• Androgens
• Corticosteroids
• Retinoids
• Thyroid hormone
• Vitamin D
• Ecdysone
• Fatty acids
• Prostaglandins

Question 18

What is the importance of the Mediator complex in transcription?

Answer 18

It facilitates the interaction between transcription factors and RNA polymerase.

Question 19

What does histone acetylation promote?

Answer 19

Nucleosome assembly and gene activation.

Question 20

What are the three main processing events in eukaryotic mRNA processing?

Answer 20

5’ capping, 3’ cleavage and polyadenylation, Pre-mRNA splicing

These events are crucial for the maturation and functionality of mRNA.

Question 21

What is the role of 5’ capping in mRNA processing?

Answer 21

Capping protects the 5’ end of RNA to make it more stable (has a free phosphate)

• Increases stability of mRNA
• Required for efficient splicing
• Linked to transcription initiation
• Nuclear export (gets RNA out of nucleus) and Translation initiation

The cap structure is added to the 5’ end of the mRNA and is essential for its stability and translation.

Question 22

What is the significance of 3’ cleavage and polyadenylation in mRNA processing?

Answer 22

PolyA tailing functionally linked to transcription (transcription termination to be specific)
Major role in stability (proportional to length of PolyA tail) and translation efficiency

Polyadenylation involves adding a polyA tail to the 3’ end of mRNA, which protects it from degradation.

Question 23

What are the signals for cleavage and polyadenylation?

Answer 23

AAUAAA, G/U or U rich region

(Part of the last exon)

These signals are recognized by specific protein factors that facilitate the cleavage and addition of the polyA tail.

Question 24

What is the first step in the splicing mechanism?

Answer 24

Cleavage at 5’ splice site (cut after the first exon) and lariat formation at Branchpoint sequence

5’ end of intron is joined onto the 2’ hydroxyl in the ‘A’ (at intron) at the end of the branch point

The lariat structure is formed as part of the splicing process, which helps remove introns from pre-mRNA.

Question 25

What are the components of the spliceosome?

Answer 25

Small nuclear ribonucleoprotein particles (snRNPs), U1, U2, U4, U5, U6 ## Footnote snRNPs are critical for the splicing process, as they form the spliceosome that catalyzes intron removal.

Question 26

What is alternative splicing?

Answer 26

- Exon inclusion/exclusion, producing proteins with altered sequences (generates many variants that are still related) or truncated proteins (mutated version) if included exon has a stop codon (may not function correctly) E.g. Sex determination in fruit flies, control of flowering time etc - Different proteins (isoforms) can be produced from a single gene - Isoforms may have different functions ## Footnote Alternative splicing allows a single gene to produce multiple protein isoforms with different functions.

Question 27

True or False: Splicing requires catalytic RNA.

Answer 27

True ## Footnote Catalytic RNA plays a vital role in the splicing process, facilitating the chemical reactions necessary for intron removal.

Question 28

What is the function of snRNPs in splicing?

Answer 28

Recognize splice sites and catalyze the splicing reaction ## Footnote snRNPs are essential for assembling the spliceosome and executing the splicing of pre-mRNA.

Question 29

Fill in the blank: The 5' cap is added to mRNA via a _______ linkage.

Answer 29

5' to 5' via the phosphates ## Footnote This unique linkage is critical for mRNA stability and function.

Question 30

What is the purpose of exon ligation during splicing?

Answer 30

To join exons together after introns are removed ## Footnote Ligation ensures that the mature mRNA contains only the coding sequences necessary for translation.

Question 31

What are the conserved sequences recognized during splicing?

Answer 31

5’ splice site, 3’ splice site, branchpoint region ## Footnote These sequences are crucial for the accurate removal of introns and joining of exons.

Question 32

List the four factors involved in cleavage and polyadenylation.

Answer 32

* Cleavage and Polyadenylation specificity Factor (CPSF) * Cleavage stimulatory factor (CstF) * Cleaving factors * Poly-A polymerase (PAP) ## Footnote These factors work together to ensure proper cleavage and addition of the polyA tail to pre-mRNA.

Question 33

What is the role of RNA-protein and RNA-RNA interactions in RNA processing?

Answer 33

Facilitate assembly of processing complexes and splicing ## Footnote These interactions are essential for the coordination and efficiency of RNA processing events.

Question 34

Describe the enhancer in the A class II regulatory region

Answer 34

- loading pad for proteins - position and orientation independent - 0 to 10 kb from promoter

Question 35

Describe the promoter in the A class II regulatory region

Answer 35

- is the TATA box - multiple binding sites for sequence-specific transcription factors - co-operate and interact with polymerase

Question 36

What proteins are involved in transcriptional stimulation in eukaryotes?

Answer 36

- General factors/ TFIID etc - RNA polymerase - Histone acetylase (HAT) = histones lose affinity to DNA - Mediator complex - Adaptors - ATP-dependent chromatin modification complexes

Question 37

Describe the general mechanism of signal transduction pathways which result in phosphorylation of transcription factors

Answer 37

*Transduction = to switch on/off a gene* - Ligand-receptor (stimulated i.e. by a hormone) interaction at plasma membrane - Generation of 2nd messenger small molecule (e.g. cAMP) - Activation of protein kinases (enzyme that adds phosphate to protein) - Regulation of transcription factor activity - Charge, structure, protein-protein interactions - DNA binding, activation, nuclear localisation, degradation

Question 38

Describe gene activation in response to viral infection

Answer 38

- ligand binds to receptor - causes activation of transcription factor - which binds to enhancer - and activates transcription

Question 39

Describe Activation of transcription by signal transduction (5 marks)

Answer 39

- Hormone receptor - ATP converted into cAMP - cAMP activates protein kinase A - protein kinase A phosphorylates CREB (a transcription factor) - CREB recruits CBP (causes histone acetylation) *CREB (cyclic AMP regulatory element binding factor) = gene activation by cyclic AMP* *CBP (CREB-binding protein) = histone acetylase that can access and acetylate chromatin, necessary but not sufficient to allow RNA polymerase to start transcribing genes*

Question 40

What are the steps in 3’ cleavage and polyadenylation?

Answer 40

1. Cleavage and polyadenylation specificity factor (CPSF) binds to AAUAA 2. Cleavage stimulatory factor (CstF) binds to G/U- recruits cleaving factors and poly-A polymerase (PAP) 3. Cleavage occurs (RNA is chopped) and addition of PolyA (adds ‘A’s onto the end)

Question 41

What are the signals for splicing?

Answer 41

Conserved sequences at the: - 5’ splice site (AGGU) - 3’ splice site (YAGGU) - branch point region (YNYURAY) = 15-30 bases from the left Recognised by factors which remove introns *R = purine (A & U)* *Y = pyrimidine (C & U)* *N = any nucleotide*

Question 42

What is the second step of the splicing mechanism?

Answer 42

Cleavage at the 3’ splice site (intron cut at the beginning of next exon) Removal of intron region and exon ligation by 5’ phosphate of exon 2 and 3’ phosphate of exon 1

Question 43

Describe the spliceosome assembly pathway

Answer 43

- Splicing carried out by small nuclear ribonucleoprotein particles (snRNPs or snurps) and protein splicing factors - snRNP = small nuclear RNA (snRNA) + numerous proteins = compose the spliceosome - snRNPs involved in splicing are U1 (identifies intron/exon boundary), U2 (recognises branch point), U4, U5 and U6 (a complex recruited by U1 and U2) - Stepwise assembly to form a spliceosome - U4, U5 and U6 complex binds to mRNA and rearranges so that U1 is on the 5’ end and U4 is displaced out of the complex **U refers to the identity of the RNA** *U3 is not involved in splicing* - U2, U5 and U6 = the active site of the spliceosome = exons are brought together and intron is looped out so it can be cut and the exons can be joined