Mod 5- Gene Expression In Eukaryotes Flashcards
What is the primary role of transcription factors?
To regulate gene expression by binding to specific DNA sequences.
What is the structure of eukaryotic DNA organized into?
Chromatin.
What does DNase hypersensitivity indicate regarding nucleosomes?
Nucleosomes are cleared from active chromatin through sliding or eviction.
What are the components of histone structure?
N-terminal variable basic arm, non-polar globular domain, C-terminal variable basic arm.
What is the significance of post-translational modifications of histones?
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
Fill in the blank: A class II regulatory region includes a _______.
[TATA box, enhancer (landing pad for proteins), promoter]
What are the two domains of a bipartite transcription factor?
Transcription activation domain - interacts with polymerase to activate transcription
DNA binding domain.
What types of interactions are involved in transcriptional stimulation in eukaryotes?
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.
What is the role of signal transduction pathways in transcriptional regulation?
They result in phosphorylation of transcription factors, modulating their activity.
True or False: Ligand-receptor interaction at the plasma membrane does not affect transcription factor activity.
False.
What are nuclear hormone receptors?
A large superfamily of ligand-dependent transcription factors/receptors.
Activated by hydrophobic molecules
What happens when a hydrophobic steroid hormone binds to its receptor?
The receptor translocates to the nucleus to activate transcription.
Fill in the blank: _______ is an example of a second messenger in signal transduction pathways.
[cAMP]
What is the bipartite nature of sequence-specific transcription factors?
They have a DNA binding domain and a transcription activation domain.
What must be modified for transcription factor access?
Chromatin structure.
What types of interactions are crucial for gene regulation?
DNA-protein interactions, protein-protein interactions, co-activators/co-repressors.
What are some examples of ligands for nuclear hormone receptors?
- Estrogens
- Androgens
- Corticosteroids
- Retinoids
- Thyroid hormone
- Vitamin D
- Ecdysone
- Fatty acids
- Prostaglandins
What is the importance of the Mediator complex in transcription?
It facilitates the interaction between transcription factors and RNA polymerase.
What does histone acetylation promote?
Nucleosome assembly and gene activation.
What are the three main processing events in eukaryotic mRNA processing?
5’ capping, 3’ cleavage and polyadenylation, Pre-mRNA splicing
These events are crucial for the maturation and functionality of mRNA.
What is the role of 5’ capping in mRNA processing?
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.
What is the significance of 3’ cleavage and polyadenylation in mRNA processing?
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.
What are the signals for cleavage and polyadenylation?
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.
What is the first step in the splicing mechanism?
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.
What are the components of the spliceosome?
Small nuclear ribonucleoprotein particles (snRNPs), U1, U2, U4, U5, U6
snRNPs are critical for the splicing process, as they form the spliceosome that catalyzes intron removal.
What is alternative splicing?
- 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
Alternative splicing allows a single gene to produce multiple protein isoforms with different functions.
True or False: Splicing requires catalytic RNA.
True
Catalytic RNA plays a vital role in the splicing process, facilitating the chemical reactions necessary for intron removal.
What is the function of snRNPs in splicing?
Recognize splice sites and catalyze the splicing reaction
snRNPs are essential for assembling the spliceosome and executing the splicing of pre-mRNA.
Fill in the blank: The 5’ cap is added to mRNA via a _______ linkage.
5’ to 5’ via the phosphates
This unique linkage is critical for mRNA stability and function.
What is the purpose of exon ligation during splicing?
To join exons together after introns are removed
Ligation ensures that the mature mRNA contains only the coding sequences necessary for translation.
What are the conserved sequences recognized during splicing?
5’ splice site, 3’ splice site, branchpoint region
These sequences are crucial for the accurate removal of introns and joining of exons.
List the four factors involved in cleavage and polyadenylation.
- Cleavage and Polyadenylation specificity Factor (CPSF)
- Cleavage stimulatory factor (CstF)
- Cleaving factors
- Poly-A polymerase (PAP)
These factors work together to ensure proper cleavage and addition of the polyA tail to pre-mRNA.
What is the role of RNA-protein and RNA-RNA interactions in RNA processing?
Facilitate assembly of processing complexes and splicing
These interactions are essential for the coordination and efficiency of RNA processing events.
Describe the enhancer in the A class II regulatory region
- loading pad for proteins
- position and orientation independent
- 0 to 10 kb from promoter
Describe the promoter in the A class II regulatory region
- is the TATA box
- multiple binding sites for sequence-specific transcription factors
- co-operate and interact with polymerase
What proteins are involved in transcriptional stimulation in eukaryotes?
- General factors/ TFIID etc
- RNA polymerase
- Histone acetylase (HAT) = histones lose affinity to DNA
- Mediator complex
- Adaptors
- ATP-dependent chromatin modification complexes
Describe the general mechanism of signal transduction pathways which result in phosphorylation of transcription factors
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
Describe gene activation in response to viral infection
- ligand binds to receptor
- causes activation of transcription factor
- which binds to enhancer
- and activates transcription
Describe Activation of transcription by signal transduction (5 marks)
- 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
What are the steps in 3’ cleavage and polyadenylation?
- Cleavage and polyadenylation specificity factor (CPSF) binds to AAUAA
- Cleavage stimulatory factor (CstF) binds to G/U- recruits cleaving factors and poly-A polymerase (PAP)
- Cleavage occurs (RNA is chopped) and addition of PolyA (adds ‘A’s onto the end)
What are the signals for splicing?
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
What is the second step of the splicing mechanism?
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
Describe the spliceosome assembly pathway
- 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
