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BMD211-Human Molecular Biology > Lecture 8: Control of Gene Expression in Eukaryotes: Part I > Flashcards

Lecture 8: Control of Gene Expression in Eukaryotes: Part I Flashcards

- DNA regulatory elements - Regulatory proteins, domain organisation and DNA -binding structures

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1
Q

Describe when eukaryotic gene expression is controlled

A

level of initiation of transcription

  • by affecting either:
  • the formation of the PIC
  • regulating local chromatin structure
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2
Q

Describe how regulatory regions where first identified

A
  • Regulatory elements in promoters were identified -> systematic replacement of short DNA segments with a DNA linker containing a random sequence of exactly the same size
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3
Q

Explain the expriment to identify regulatory regions

A
  • Overlapping linker scanning mutations (rectangles) were performed from one end of the region under investigation to the other
  • Each rectangle =position in which a linker replaced a 6-10 nucleotide segment
  • Experiment shows-> thymidine kinase gene transcription is blocked by mutations in 3 distinct regions/sequence motifs that are just upstream from the transcription initiation site
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4
Q

Explain what the proximal promoter

A
  • CCAAT box (GGCCAATCT) +GC box (GGGCGG) are present in the promoters of many eukaryotic genes
    + are between 50 and 200 bp upstream from the transcription initiation site
  • The region just upstream from the core promoter =proximal promoter
  • CCAAT and GC boxes are promoter proximal elements (PPEs)
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5
Q

Explain what enhancers

A
  • Enhancer regulatory sequences stimulate transcription
  • SV40 PPE consists of six GC boxes (GGGCGG)
  • Enhancer is in between 116-261 upstream of the transcriptional start site
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6
Q

Explain the properties of enhancers

A
  • Act at a distance
  • Orientation-independent
     Position can be upstream or downstream of the transcription initiation site
  • Can be cell-type- or tissue-specific -> active in one cell-type/tissue but NOT in another
  • Activator (protein) binds to enhancer (DNA)
  • Cell must have transcription activator proteins capable of binding to enhancer-> for full gene expression to occur
  • Activator proteins can act in a no. of different ways to turn on gene expression
     including directly interacting with the PIC + promoting open chromatin structure/euchromatin
  • Can be within transcription unit
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7
Q

Describe how activators can act on enhancers

A
  • Activator proteins can act in a no. of different ways to turn on gene expression
     including directly interacting with the PIC + promoting open chromatin structure/euchromatin
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8
Q

Describe what silencers and repressors are

A
  • Eukaryotes have negative regulatory elements -silencers
     Silencers: sequence specific DNA elements that repress transcription of a target gene
  • Mostly function independently of distance and orientation from/to target gene
  • Transcriptional repressors: DNA binding protein that binds to silencer
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9
Q

Explain how silencers can act to repress transcription

A
  • Silencers act by:
  • > establishing repressive chromatin /heterochromatin
  • > prevent nearby transcriptional activators from binding to its binding site
  • > blocking PIC formation
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10
Q

List what is contained in a higher mammalian gene

A

Mammalian genes contain:

  • promoter proximal elements
  • enhancers
  • silencers
  • core promoter
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11
Q

List the regulatory elements and describe how they can regulate gene expression

A
  • Enhancers, silencers and PPEs consist of clusters of modules (DNA sequence motifs)
    -> binds specific transcription activator or transcriptional repressor proteins
    = provides a mechanism for gene regulation ->by controlling the amount of functional transcription activator or repressor proteins with the cell nucleus
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12
Q

Explain how activators and depressors interact with promoter elements

A
  • Transcription activator/repressor proteins have at least 2 independently folded + distinct functional domains:
     DNA-binding domain -> makes sequence specific contacts with the control elements in the enhancer
     Activation or repression domain is left ‘free’ -> recruits/binds various components of the transcription machinery OR to alter chromatin structure around the transcriptional start site
    = to activate transcription
     Additional domains include: Dimerization domains and ligand binding domains
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13
Q

Describe EMSA

A
  • Used to determine protein-DNA binding
  • Label DNA with radioisotope + add protein or fractions of nuclear extract
  • Migration of DNA fragment is slow -> when bound to protein
    =causing a shift in the location of the radiolabelled DNA fragment
  • Visualised by autoradiography on X-ray film
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14
Q

Explain how EMSA is used to identify recognition site

A
  • Used to determine the exact position/sequence a protein binds to DNA
  • Label DNA with radioisotope and add protein or fractions of nuclear extract
  • Protein bound to DNA protects that region from digestion by a nuclease
  • Region of DNA protected by the bound protein
  • > the binding site appears as a gap or “footprint” in the array of bands
  • Visualised by autoradiography on X-ray film after electrophoresis
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15
Q

Explain how cell-based assay identifies activators

A
  • Determine the type of activity of a DNA- binding protein that regulates transcription
  • Activators or repressors ability to activate or repress transcription can be determined-> w/ in an in vivo transfection assay
  • System requires 2 plasmids:
  • > one containing the potential transcriptional activator or repressor (red)
  • > other contains a reporter gene (orange) + one or more binding sites for potential activator/repressor
  • Both plasmids are transfected into cells at the same time
  • Production of the reporter gene mRNA (and protein) is measured
  • The reporter gene -> often encodes green fluorescent protein = easy to determine if activator or repressor
  • Useful for using domains/truncated proteins to identify +map activator/repressor domains
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16
Q

Give the example used for identifying activators

A
  • GAL4 =yeast transcriptional activator
  • > binds to UAS sequences
  • Use of deletion mutants-> identified both the DNA binding domain (aa 1-74) +the activation domain (aa 738-823)
  • DNA binding regions are determined by EMSA
  • Activation regions by cell-based reporter assay
17
Q

Explain the structure of homeoboxes and its fucntion

A
  • Homeobox proteins contain a DNA-binding region called the homeodomain
  • The homeodomain contains a helix-turn-helix motif
  • ->highly conserved between different homeodomain-containing proteins
  • The recognition helix region binds in the major groove of DNA by a.a and mediates sequence-specific binding
  • Homeodomain found in transcription regulatory proteins
  • Regulate many important developmental genes
18
Q

Explain the structure of zinc fingers

A
  • Many eukaryotic proteins in secondary structure have regions that fold around a central Zn2+ ion -> form tertiary structure
  • A pair of cysteines and a pair of histidines act together to bind a zinc ion->causing a fold known as a zinc finger
  • This C2H2 zinc finger is the most common DNA-binding motif encoded in the human genome
  • 23- to 26- amino acid consensus
  • Many proteins 1+ ZF
19
Q

Explain the structure of zinc fingers

A
  • Many eukaryotic proteins in secondary structure have regions that fold around a central Zn2+ ion -> form tertiary structure
  • A pair of cysteines and a pair of histidines act together to bind a zinc ion->causing a fold known as a zinc finger
  • This C2H2 zinc finger is the most common DNA-binding motif encoded in the human genome
  • 23- to 26- amino acid consensus
  • Many proteins 1+ ZF
20
Q

Explain the folding of proteins in their secondary structure to form zinc fingers

A
  • The finger region =2 antiparallel β– sheets+ followed by an α–helix with the zinc ion buried in the interior
  • Zinc ion binds with tetrahedral geometry to Cys-3 and Cys-6 in the β–strand and to His19 and His-23 in the α-helix
21
Q

Explain how proteins in zinc fingers interact with DNA/RNA and other proteins

A
  • ZF present in transcription factors
  • > regions outside of ZF interact with DNA/RNA/ other proteins
  • 3 zinc finger protein interacts with three 3bp sub-sites on DNA as the zinc-finger regions wraps around one turn of the DNA helix
  • Overall binding specificity and affinity are determined by contributions from all three zinc fingers
22
Q

Explain how hormone dependent nuclear receptors act

A
  • In absence of hormone ->the nuclear receptor is kept in the cytoplasm through an interaction with inhibitors
  • In presence of hormone->diffuses through plasma membrane and binds to LBD (ligand binding domain)
    = releasing inhibitor
  • Then nuclear receptor moves to nucleus + binds as a dimer (homo- or hetero-) to response elements -> through its zinc finger DNA binding domain (DBD)
23
Q

State what parts of the nuclear receptor is conserved

A
  • Highly conserved (DBD) +conserved LBD

- Activation function domains (AF1 and 2) and variable hinge region and are not conserved

24
Q

Explain how nuclear receptor domain swapping shows function

A
  • Effects of swapping to have ER with DBD of GR

- Chimera causes-> binding of estrogen to ER + induces transcription of glucocorticoid responsive gene

25
Q

Explain the binding of GR to DNA

A
  • GR binds to DNA as a homodimer
  • Contact DNA bases in the major groove on the same side of the DNA molecule
  • GR binds to glucocorticoid response element (GRE)
  • GRE has two 6-bp palindromic half sites separated by three nucleotides
26
Q

Explain how leu zippers are formed

A
  • Found in some proteins that contained a leucine every 7th amino acid over a 35 amino acid region
  • Predicted to form an α-helix in which Leu would occur every 2 turns (α-helix has 3.6 amino acids per turn)
    = Leu side chains occur on one face
  • α-helices segments that are either identical (homo) or different dimerise-> used to characterise proteins
  • Leu side chains interdigitate like teeth on a zipper
27
Q

Explain how Leu zipper interacts with DNA

A
  • The leucine zipper does NOT directly bind to DNA
  • Facilitates dimerisation of the protein
  • The dimerisation region often forms a coiled -coil to ‘join’ two proteins
    +facilitate subsequent DNA binding
  • The DBD is highly basic + together with the leucine zipper forms the bzip DBD
28
Q

Give example of a protein that is leu zipper and describe how it interacts with DNA

A
  • Gcn4 contains a bzip DNA binding domain

- Binds in major groove to two semi-palindromic half sites

29
Q

Give example of a proteins that can do bzip heterodimerisation and how that affects DNA binding

A
  • c-Jun can bind DNA as a homodimer or as a heterodimer with c-Fos
  • Dimerisation by the leucine zipper allows-> different complexes w/different binding affinities + different activity to form on an identical DNA-binding site
30
Q

State how activators with bHLH domains bind and what they contain

A
  • Activators with basic- helix- loop- helix domain bind to DNA as homo- or heterodimers
  • Contain a basic recognition helix and HLH dimerisation domain
31
Q

State how activators with bHLH domains bind and what they contain

A
  • Activators with basic- helix- loop- helix domain bind to DNA as homo- or heterodimers
  • Contain a basic recognition helix and HLH dimerisation domain
32
Q

Explain how Myc, Max and Mad proteins bind

A
  • Myc and max proteins can form heterodimers
  • Max and mad can form heterodimer too
  • Mad is another bHLH zip protein
33
Q

Describe the structural domains and characteristics of the activators

A
  • Homeobox: homeodomain-> DNA binding role-> in drosophila homeostatic genes-> DNA binding mediated through helix-turn-helix motif
  • Cysteine-histidine ZF-> DNA binding role-> in TFIIIA-> multiple copies of ZF motif
  • Cysteine-Cysteine ZF finger-> DNA binding role-> In nuclear receptor family-> single pair of fingers + relate motifs in GAL4 (yeast)
  • Basic element-> DNA binding role-> c-fos,c-jun-> Found in association with Leu zipper
  • Leu zipper-> protein dimerisation role-> c-jun, c-fos, c-myc-> mediates dimerisation =essentail for DNA binding by adjacent domain
  • Helix-loop-helix-> protein dimerisation role-> c-myc->
34
Q

Describe the structural domains and characteristics of the activators

A
  • Homeobox: homeodomain-> DNA binding role-> in drosophila homeostatic genes-> DNA binding mediated through helix-turn-helix motif
  • Cysteine-histidine ZF-> DNA binding role-> in TFIIIA-> multiple copies of ZF motif
  • Cysteine-Cysteine ZF finger-> DNA binding role-> In nuclear receptor family-> single pair of fingers + relate motifs in GAL4 (yeast)
  • Basic element-> DNA binding role-> c-fos,c-jun-> Found in association with Leu zipper
  • Leu zipper-> protein dimerisation role-> c-jun, c-fos, c-myc-> mediates dimerisation =essentail for DNA binding by adjacent domain
  • Helix-loop-helix-> protein dimerisation role-> c-myc-> Mediates dimerisation =essentail for DNA by adjacent domain

BMD211-Human Molecular Biology (5 decks)

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