Lecture 5: Eukaryotic genome — Transcriptional Regulation

Question 1

Why is transcriptional regulation needed?

Answer 1

• Allows development of different tissues
• Transition from childhood to adult
• Deregulation can result in uncontrolled growth (cancers)
• Allows reaction to environmental cues

Question 2

How is transcription controlled?

Answer 2

• Chromatin structure
• RNA polymerase (n general TF) binding specificity
• Additional binding n activation factors

Question 3

How does the histone code regulate chromatin structure to influence gene expression?

Answer 3

• 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”

Question 4

What is the model for the structure of an interphase chromosome?

Answer 4

• 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

Question 5

What are the 2 major types of heterochromatin?

Answer 5

• 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

Question 6

Describe the human centromere’s organization

Answer 6

• Centric heterochromatin: long highly repetitive chromatin structures
• H3K4me2: allows open structure for kinetochore attachment

Question 7

Describe the end replication problem

Answer 7

• 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

Question 8

What is the Hayflick limit?

Answer 8

• 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)

Question 9

Describe the compensatory mechanism for telomere shortening

Answer 9

• 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.

Question 10

Describe the sequence of events involved in partial lengthening of telomeres

Answer 10

• 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

Question 11

Why do telomeres not fuse despite having a free 3’ unpaired end?

Answer 11

• 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

Question 12

What is the shortening of telomeres associated with?

Answer 12

Aging

Question 13

Give an example of a disease associated with premature aging

Answer 13

• Werner Syndrome
• Lagging strand synthesis is not replicated efficiently in Werner cells
• Overexpression of telomerase in vitro counteracts WRN mutation

Question 14

What type of RNA polymerases do we have and what are their functions?

Answer 14

• 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

Question 15

What does a human Pol II promoter look like?

Answer 15

• 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

Question 16

What are the general transcription factors needed for transcription initiation by eukaryotic RNA Pol II n their functions?

Answer 16

• 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

Question 17

What are the steps involved in the assembly of the basal transcription apparatus?

Answer 17

• 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

Question 18

How does the TATA box binding protein find the TATA box?

Answer 18

• 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.

Question 19

How does RNA polymerase II transition from initiation to elongation?

Answer 19

• 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

Question 20

What are the roles of TFIIH in transcription initiation and elongation?

Answer 20

• 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.

Question 21

Describe the G-less cassette transcription assay

Answer 21

• 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

Question 22

Explain how different TATA sequences support different levels of transcription. Provide examples.

Answer 22

• 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

Question 23

How does the TATA box regulate expression in viruses?

Answer 23

• 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

Question 24

What other cis-acting elements regulate transcription in EBV?

Answer 24

• 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

Question 25

What are the 2 types of TF and what are their functions?

Answer 25

• 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

Question 26

What design do TFs have?

Answer 26

• 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.

Question 27

What are homeodomains?

Answer 27

• 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.

Question 28

What are zinc finger motifs?

Answer 28

• 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.

Question 29

What are leucine zippers?

Answer 29

• 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

Question 30

Explain multimerization n combinatorial control in terms of T-cells

Answer 30

• 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

Question 31

What is the enhanceosome?

Answer 31

• 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.

Question 32

What is an example of an enhanceosome?

Answer 32

• 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

Question 33

What is the Philadelphia chromosome?

Answer 33

• 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.

Question 34

What is Burkitt’s lymphoma?

Answer 34

• 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