Lecture 5: Eukaryotic genome — Transcriptional Regulation Flashcards
1
Q
Why is transcriptional regulation needed?
A
- Allows development of different tissues
- Transition from childhood to adult
- Deregulation can result in uncontrolled growth (cancers)
- Allows reaction to environmental cues
2
Q
How is transcription controlled?
A
- Chromatin structure
- RNA polymerase (n general TF) binding specificity
- Additional binding n activation factors
3
Q
How does the histone code regulate chromatin structure to influence gene expression?
A
- Open chromatin or close it into a condensed form, shifting the balance between expression and silencing
- Operated by the histone code with activation opening the DNA or condensing the chromatin and silencing making for heterochromia that is not normally expressed.
- Activation: expressed genes are found in “open”
4
Q
What is the model for the structure of an interphase chromosome?
A
- Compact 30 nanometer fiber
- Non-histone protein chromosome scaffold (poorly understood)
- Rich in topoisomerases that regulate torsional changes caused by packing/unpacking
- Maybe loops extend where we need to express them
5
Q
What are the 2 major types of heterochromatin?
A
- Facultative
- Cell-type specific
- Can switch into euchromatin following developmental cues
- Characterized by a specific histone code mark (H3K27me3) that binds “polycomb” proteins
- Constitutive
- Consistently silenced in all cell types of an organism
- Centromeres
- Telomeres
- Transposon
- Characterized by H3K9me3 (modification carried out by histone methyltransferase HMT)
- HMT propagate heterochromatin by recognizing H3K9me3 n methylating adjacent nucleosomes
- Consistently silenced in all cell types of an organism
6
Q
Describe the human centromere’s organization
A
- Centric heterochromatin: long highly repetitive chromatin structures
- H3K4me2: allows open structure for kinetochore attachment
7
Q
Describe the end replication problem
A
- Because DNA synthesis can only proceed 5’ to 3’ there is continuous synthesis on the leading strand and discontinuous synthesis on the lagging strand: synthesis requires RNA primers
- The lagging strand template can be primed near the telomere (and then extended
- DNA polymerase complex disengages
- RNA primers are erased
- Gap is filled by DNA polymerase n repaired by DNA ligase
- Gap on the lagging strand cannot be filled by a DNA polymerase [no primer thus no 3’ - OH for extension]
- EFFECT: telomeres shorten after each cell division
8
Q
What is the Hayflick limit?
A
- Number of times a normal somatic, differentiated human cell population will divide before cell division stops (b/w 40-60 divisions)
- RESULT: cell becomes senescent (in built aging mechanism)
9
Q
Describe the compensatory mechanism for telomere shortening
A
- Telomerase binds the single-stranded G-overhang: a ribonucleoprotein (RNP) enzyme made of the telomerase RNA (TER) and telomerase reverse transcriptase protein (TERT).
- It extends the 3’ end of the parental strand using its own RNA subunit as a template.
10
Q
Describe the sequence of events involved in partial lengthening of telomeres
A
- RNA-templated DNA synthesis by telomerase extends the G-overhang 5’-3’ DNA primase lays down an RNA primer on the extended G-overhang
- DNA-templated DNA synthesis by DNA Polymerase extends this primer 5’-3’ DNA ligase ligates the new Okazaki fragment to the old lagging strand 5’ end
- There is still a free 3’ unpaired end that triggers repair mechanisms
11
Q
Why do telomeres not fuse despite having a free 3’ unpaired end?
A
- A shelterin complex of
- TRF1 (telomeric repeat-binding factor 1)
- TRF2 (telomeric repeat-binding factor 2)
- RAP1 (repressor/activator protein) and others
- Stimulates t-loop formation
- That displaces a d-loop and results in the base pairing of the 3’ end.
- RESULT: The 3’ end shelters from repair mechanisms in a telosome
12
Q
What is the shortening of telomeres associated with?
A
Aging
13
Q
Give an example of a disease associated with premature aging
A
- Werner Syndrome
- Lagging strand synthesis is not replicated efficiently in Werner cells
- Overexpression of telomerase in vitro counteracts WRN mutation
14
Q
What type of RNA polymerases do we have and what are their functions?
A
- RNA Pol I: produced rRNA
- RNA Pol II: all protein coding genes
- RNA Pol III: Transcribes genes for specific types of RNA involved in gene regulation
15
Q
What does a human Pol II promoter look like?
A
- TATA box required for polymerase transcription
- Binds to binding protein or TBP (TATA binding protein)
- 1+ for transcription
- Downstream
- Split where eukaryotic gene followed by an encoded poly signal
- Transcription terminator
16
Q
What are the general transcription factors needed for transcription initiation by eukaryotic RNA Pol II n their functions?
A
- TFIID
- TBP
- TAF
- FUNCTION: recognizes TATA box, regulates DNA binding
- TFIIB
- Positions RNA Pol I over start site
- TFIIF
- Binds to RNA Pol II → stabilizes it
- TFIIE
- Regulates TFIIH
- TFIIH
- Unwinds DNA n phosphorylates Ser5 domain of RNA Pol II → activates it
17
Q
What are the steps involved in the assembly of the basal transcription apparatus?
A
- TBP part of TFIID binds to TATA box
- Further subunits are recruited
- TFIID complex binds to TATA box via TBP aided by TFIIA
- TBP recruits TFIIB which recognizes BREu n BREd
- Positions RNA Pol II at the start of transcription site (+1)
- TFIIE, RNA Pol II/TFIIF recruited
- TFIIF stabilizes RNA Pol II interactions w TFIIE n TFIIH
- TFIIH recruited by TFIIE
18
Q
How does the TATA box binding protein find the TATA box?
A
- TATA box is a consensus sequence
- Individual TATA boxes have different affinities for TBP – and so some are more efficient at stimulating transcription than others.
19
Q
How does RNA polymerase II transition from initiation to elongation?
A
- RNA polymerases do not require a primer. Elongation starts.
- RNA Pol II disengages from transcription factor cluster → conformational change that tightens its interaction with DNA.
- Phosphorylation of the CTD marks the transition from initiation to elongation
20
Q
What are the roles of TFIIH in transcription initiation and elongation?
A
- TFIIH is a complex of proteins.
- Helicase that opens the DNA double helix → polymerase to accesses template strand
- Kinase that phosphorylates the C’-terminal domain (CTD) of the RNA Pol II L’ subunit
- How can polymerase binding to the TATA box regulate transcription?
- Individual TATA boxes have different affinities for TBP – and so some are more efficient at stimulating transcription than others.
21
Q
Describe the G-less cassette transcription assay
A
- G-less cassette is an artificial piece of DNA made that lacks G residues
- Promoter is cloned upstream of G-les cassette
- Purified TFs n RNA Pol II, ATP, CTP n [α32P]-UTP (radioactive) are added
- RNA is truncated at the point of which a G should be inserted [no GTP supplied]
- RESULT: radioactive RNA transcript of a defined size (typically 400 bp)
- Can be electrophoresed thru polyacrylamide gels n quantified following autoradiography
22
Q
Explain how different TATA sequences support different levels of transcription. Provide examples.
A
- The major late adenovirus promoter (AdML) is a (human) viral promoter (TATAAAA)
- HeLa (human) TFIID binds strongly and supports high expression of the G-less cassette.
- TATAAAG. Human TFIID binds less strongly to the yeast His TATA box → reduced expression.
- No obvious TATA box → no expression of the G-less cassette
23
Q
How does the TATA box regulate expression in viruses?
A
- EXAMPLE: Epstein-Barr virus (EBV)
- EBV DNA encodes 2 IE mRNA
- IE: immediate early
- IE proteins makes TF that act upon the DNA n allow gene expression
- SEQUENCE: TATA
- Some early proteins stimulate DNA replication → late genes
- SEQUENCE: TATT
- Temporal control: the TATA box sequence changes depending on when the gene n at what time the gene is expressed
24
Q
What other cis-acting elements regulate transcription in EBV?
A
- IE gene
- TATA box w proximal positive cis-acting elements enhance transcription n both proximal n distal negative cis-acting elements that inhibit transcription
- E gene
- TATA box w both proximal n distal positive cis-acting elements that enhance transcription
- L gene
- TATT version of TATA box
25
Q
What are the 2 types of TF and what are their functions?
A
- General TF
- Assemble promoter n form complex w RNA Pol II
- Specific TF
- Activators → increase transcription
- Repressors → decrease transcription
- These TF bind to the proximal promoter n distal (enhancer) elements
26
Q
What design do TFs have?
A
- DNA-binding domain that binds specific DNA sequences
- Activating/repressing domain (protein interaction domain) that stimulates/inhibits transcription by interacting with mediator proteins, general transcription factors or RNA Pol II.
27
Q
What are homeodomains?
A
- DNA binding domain that defines a class of gene regulatory proteins
- Helix 3 binds in the major groove of DNA making specific interactions between amino acids and nucleotides.
28
Q
What are zinc finger motifs?
A
- 2 β strands
- 2 cystine residues in β sheet bind to 1 Zn molecule
- 1 α helix
- 2 histidine residues bind to the same Zn molecule
- 4 amino acids coordinate Zn molecule → stabilizes fold
- Often there is cluster, arranged one after the other so that the α-helix of each binds the major groove of the DNA.
- A strong and specific DNA-protein interaction is built up through a repeatingbasicstructural unit.
29
Q
What are leucine zippers?
A
- Each of the DNA binding domain of the leucine zipper binds to a symmetrical DNA sequence that’s palindromic
- Cross-sheet binding to specific sequences
- Can also bind to DNA as heterodimers, expanding potential regulatory repertoire
- EXAMPLE: Jun/Fos heterodimer
- Can exist as both hetero n homodimers
- Combination of subunits leads to regulation n control
30
Q
Explain multimerization n combinatorial control in terms of T-cells
A
- Jun n Fos heterodimer combines n binds to target site in T cells (ATP1 complex) → initiates low lvl of expression in IL-2
- NFAT (nuclear factor of activated T-cells) binds to different motif nearby → low lvl expression of IL-2
- All 3 pathways activated simultaneously → high lvl expression of IL-2
31
Q
What is the enhanceosome?
A
- In genes that require tight control, activators bind cooperatively along an enhancer sequence → enhanceosome.
- Each enhanceosome is unique to its specific enhancer.
- FUNCTION
- Recruitscoactivatorsandgeneral transcription factorsto thepromoter regionof the target gene to begin transcription
- Recruits non histone architectural transcription factors (high-mobility group, HMG) proteins, which regulate chromatinstructure – they ensure that the target gene can be accessed by transcription factors.
32
Q
What is an example of an enhanceosome?
A
- NF-κB, interferon activator proteins, and theATF-2/c-Jun complex - cooperatively bind the enhancer upstream of the humaninterferon-βgene
- Enhanceosome recruits HMG-1 and the transcriptional machinery to the promoter → initiates high levelgene expression of IFN-β
33
Q
What is the Philadelphia chromosome?
A
- Gene translocation effect b/w chromosome 9 n 22
- Brings powerful cell division kinase ABL to the break cluster region (BCR)
- RESULT: brings BCR chromosome n enhancer next to a gene fusion of BCR N ABL
- BCR-ABL1 protein lacks the first exon of ABL1 → ABL1 tyrosine kinase activity permanently ON,
- TK activity of ABL is now under the control of the BCR promoter/enhancer: unregulated expression of an oncogene → cell proliferation
- All cases of chronic myeloid leukaemia (CML) carry a Philadelphia re-arrangement.
34
Q
What is Burkitt’s lymphoma?
A
- Translocation b/w chromosome 8 n 14
- Bringing the MYC gene (which regulates cell division under the control of immunoglobulin promoter)
- RESULT: unregulated cell proliferation, often accompanied by gene instability
