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5BBG0205 Molecular Basis of Gene Expression > L3: Eukaryotic Transcription Factors > Flashcards

L3: Eukaryotic Transcription Factors Flashcards

1
Q

What are transcription factors (TFs) and their main types?

A
  • TFs are proteins required for initiating/regulating transcription.
  • Two main types:
    1. General factors (form basal transcription complex)
    2. specific factors (bind regulatory sequences)
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2
Q

How do transcription factors bind to DNA?

A
  • TFs bind to DNA through specific interactions between AAs & base pairs.
  • DNA-protein interface involves 10-20 contacts between AAs and bases.
  • Asparagine & arginine side chains interact with DNA’s outer sugar-phosphate backbone
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3
Q

What are promoters and where are they located?

A
  • Promoters = DNA sequences that initiate & regulate transcription
  • Mainly located upstream (5’) of the transcribed sequence.
  • Close to transcription start site
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4
Q

What is the structure of a typical Pol II promoter?

A

Regulatory promoter:
GC box at -100 bp.
CCAAT sequence at -80 bp.
GC box at -50 bp.
Core promoter:
TATA (or Hogness) box at -20 bp.

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5
Q

What are enhancers and silencers, and where are they located?

A
  • Enhancers/silencers = distant DNA sequences (thousands of base pairs) from transcription start site.
    Can be located upstream or downstream, up to 50 kbp away.
    Can range from 50 bp to 1.5 kbp in size.
    Contain multiple binding sites for different TFs
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6
Q

How do enhancers function and interact with the transcription initiation complex?

A
  • Enhancer binding sites bind activator proteins
  • Can indirectly initiate transcription by binding transcription initiation complex via a mediator molecule.
  • DNA flexibility allows looping to facilitate interactions
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7
Q

What are insulators and their role?

A
  • Insulators prevent interference between transcription units.
  • Demarcate zone of influence of regulatory sequences on DNA
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8
Q

How do specific transcription factors interact with the transcription initiation complex?

A
  • directly or indirectly
  • Gene regulatory proteins bind specific regulatory sequences in the spacer DNA, located upstream of promoter region
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9
Q

What is the role of general transcription factors in the initiation of transcription?

A
  • GTFs directly bind to the promoter region of DNA
  • this enables binding of RNA polymerase II & initiates transcription
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10
Q

What are housekeeping genes?

A
  • Housekeeping genes = expressed in all cells (they’re required for maintenance of basic cellular function)
    Examples: Histone genes coding for chromatin structural proteins
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11
Q

How do promoters of housekeeping genes facilitate efficient transcription?

A
  • Promoters of housekeeping genes have binding sites for ubiquitous TFs present in all tissues
  • this increases the efficiency of transcription for these genes.
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12
Q

What are some common consensus sequences in promoters and their positions?

A
  • CAAT box: GGCCAATCT, usually at -80 position.
  • GC box: GGGCGG, often at -40 to -80 position.
  • Octamer: ATTTGCAT, variable position.
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13
Q

What are tissue-specific genes?

A
  • Tissue-specific genes have binding sites for both ubiquitous & tissue-specific TFs
  • Examples incl. globin genes with binding sites for Sp1, CP1 (ubiquitous), and GATA-1 (tissue-specific)
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14
Q

What are the control regions in the β-globin gene and what do they contain?

A

β-globin gene has a 5’ control region and a 3’ control region
These regions contain various binding sites including the TATA box

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15
Q

How can binding sites interact with different types of transcription factors?

A

Same binding sites can bind either ubiquitous or tissue-specific factors based on context (sequences, cell type, TFs present)

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16
Q

What are inducible genes?

A
  • Inducible genes are expressed in response to specific signals/conditions.
  • they contain binding sites for inducible transcription factors.
  • Example: Growth hormone gene with binding sites for Pit-1, glucocorticoid receptor (GR), and CREB
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17
Q

How does the Pit-1 transcription factor affect gene expression?

A
  • Pit-1 is an anterior pituitary-specific TF
  • Mutation in Pit-1 leads to dwarf phenotype in mice
  • It binds to specific binding sites in the growth hormone gene promoter
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18
Q

What are foot printing techniques used for?

A
  • used to detect transcription factor binding to DNA
  • they identify specific DNA sequences recognized & bound by TFs
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19
Q

Foot printing techniques are used to detect transcription factor binding to DNA.
They identify specific DNA sequences recognized and bound by transcription factors

A
  1. Multiple copies of a DNA fragment with a radioactive label are divided into two samples.
  2. Sample 1 is incubated with a protein (individual or mixture).
  3. Protein binds to a specific DNA sequence.
  4. Sample 1 is digested with DNase (endonuclease enzyme).
  5. Denaturation is carried out to expose the labeled DNA strand.
  6. Sample 2 is incubated without proteins.
  7. Sample 2 is digested with DNase.
  8. Denaturation is carried out for both samples.
  9. Electrophoresis autoradiography separates fragments by size
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20
Q

How does footprinting detect transcription factor binding sites?

A
  • only the DNA sequence bound by the protein remains protected from denaturation
  • this protected sequence will not appear in the autoradiography
  • binding site of a specific TF is thus identified
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21
Q

What is Chromatin Immunoprecipitation (ChIP) used for?

A
  • ChIP detects DNA sequences bound by specific TFs in vivo
  • it identifies which DNA sequences are bound by specific TFs within the context of the cell
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22
Q

Explain the steps of the Chromatin Immunoprecipitation (ChIP) technique

A
  1. HCHO (formaldehyde) creates cross-links between TFs and DNA sequences.
  2. DNA is sheared, resulting in fragments with specific and general TFs.
  3. Immunoprecipitation uses an antibody to bind the specific TF.
  4. Cross-links between the TF and antibody are reversed.
    Protein/TF is removed.
  5. DNA is purified and sequenced to identify the TF-bound sequence
23
Q

What is the purpose of a Reporter Gene Assay?

A
  • Reporter Gene Assay used to investigate the impact of a TF binding on the transcription of a gene.
  • It measures the effect of TF binding on the expression of a reporter gene
24
Q

How can mutation experiments be combined with the Reporter Gene Assay?

A
  • Mutation experiments involve introducing mutations into the promoter region of a gene
  • Mutations in essential DNA sequences can inhibit transcription.
  • By analyzing the effects of mutations on reporter gene expression, crucial TF binding sites can be identified
24
Q

What is Affinity Chromatography used for in transcription factor research?

A
  • Affinity Chromatography = used to purify TFs based on their specific DNA-binding properties.
  • helps identify which TFs in a cell type bind to specific DNA sequences important for transcription regulation
25
Q

What are structural motifs used for in classifying transcription factors?

A
  • Structural motifs used to classify TFs into families
  • These motifs can be DNA-binding domains or dimerization domains
25
Q

Name some common DNA-binding structural motifs of transcription factors

A

Helix-turn-helix
Zinc finger

26
Q

Name some common dimerization structural motifs of transcription factors

A

Leucine zipper
Helix-loop-helix

26
Q

How might a transcription factor have multiple domains?

A
  • TFs can have separate domains for DNA-binding & DNA-activating functions
    (In some cases, they might even have connecting domains that link these functions together)
27
Q

What is the Helix-Turn-Helix motif in transcription factors, and where is it found?

A
  • Helix-Turn-Helix (HTH) motif is a DNA-binding motif
  • found in many prokaryotic & eukaryotic TFs
28
Q

Describe the structure of the Helix-Turn-Helix motif.

A
  • The Helix-Turn-Helix motif consists of two α helices connected by an AA “turn.”
  • The C-terminal “recognition” helix makes contact with the major groove of DNA.
29
Q

Provide examples of transcription factors that have the Helix-Turn-Helix motif

A

Homeodomain proteins
POU proteins
Pax proteins

30
Q

How do homeodomain proteins contribute to embryonic development?

A
  • mutations in genes coding for homeodomains can lead to homeotic transformations = where body parts are changed during embryonic development.
  • e.g. mutations in Drosophila HOM-C genes can result in body part transformations
31
Q

Describe the structure of the Homeodomain

A
  • consists of approximately 60 AAs
  • forms 3 alpha helices, including the helix-turn-helix motif.
  • The C-terminal third alpha helix, known as the recognition helix, determines DNA-binding specificity
32
Q

What are POU proteins and how are they structured?

A
  • POU proteins = TFs with POU homeodomain & POU-specific HTH domain
  • POU homeodomain + POU-specific HTH domain are both needed for high-affinity, sequence-specific DNA binding.
  • Some POU proteins, like Oct-1, have a POU-region consisting of a POU-specific HTH domain, a linker, and a POU homeodomain
33
Q

What are Pax proteins, and how are they structured?

A
  • Pax proteins = contain a paired type of helix-turn-helix domain - with 2 helix-turn-helix motifs linked together.
  • Pax proteins may also include a homeodomain.
  • They regulate cell fate during embryonic development, such as Pax-6 for iris eye development
34
Q

What is the Zinc Finger motif, and how is it structured?

A
  • The Zinc Finger = a DNA-binding motif found in TFs
  • consists of zinc ions coordinated by cysteine and histidine residues.
  • Zinc fingers classified into Type 1 & Type 2 - each with different structures and functions
35
Q

Describe the structure of Zinc Finger Type 1

A
  • Type 1 zinc fingers have a finger loop formed by 2 cysteines and 2 histidines (or 4 cysteines) that bind a central zinc atom
  • finger loop includes an α-helix and a β-sheet
  • Multiple zinc fingers interdigitate with DNA via direct contact with alpha helices - making sequence-specific contacts with the major groove of DNA
36
Q

Give examples of transcription factors that contain Zinc Finger Type 1 motifs

A
  • Sp1 (3 fingers)
  • GATA factors (2 fingers)
  • Nuclear receptors: Group I (steroid hormone receptors), Group II (non-steroid hormone receptors)
37
Q

How do Zinc Finger motifs bind to DNA?

A
  • Zinc fingers bind various DNA sequences depending on the interaction of specific AA in zinc finger with different bases on DNA strands.
  • interaction of zinc fingers with DNA can determine binding to either leading or lagging strand/or both
  • the sequence recognized by zinc fingers is named based on the specific AAs & DNA bases involved in the binding
38
Q

How can re-engineered zinc fingers be used for gene therapy?

A
  • Re-engineered zinc fingers can recognize & bind specific genome sequences - enabling targeted gene therapy.
  • binding specificity of zinc finger to major groove of DNA can be reprogrammed by changing AAs that recognise specific bases
  • Zinc fingers can be linked to endonucleases that cleave DNA, allowing for gene knockout mutations
39
Q

Describe an example of anti-HIV gene therapy using Zinc Finger proteins

A
  • Zinc Finger proteins could target and disrupt gene encoding cell surface receptors (CCR5) that HIV uses to enter T-cells
  • by disrupting the CCR5 gene, HIV entry into T-cells would be prevented → leading to a replacement of the infected blood population with non-infected cells
40
Q

What are Type 2 Zinc Fingers, and how are they structured?

A
  • Type 2 Zinc Fingers = separate class of zinc-finger TFs found in nuclear receptors.
  • They have two Cys2/Cys2 fingers: one for DNA-binding & other for dimerization.
  • Ligands (such as steroids, retinoids, vitamin D, and thyroid hormones) interact with these receptors
41
Q

What is the structure of nuclear receptors with Type 2 Zinc Finger motifs?

A
  • Nuclear receptors have a central DNA binding domain containing zinc fingers (conserved)
  • C-terminal ligand binding domain (variable)
  • N-terminal activation domain that regulates transcription
42
Q

Describe the binding characteristics of Group 1 Steroid Hormone Nuclear Receptors

A
  • Group 1 steroid hormone nuclear receptors bind as monomers/homodimers to related 6-bp sequences on DNA.
  • sequences arranged as 2 palindromic repeats, allowing for sequence-specific binding
  • ligand binding regulates their entry into nucleus, where they regulate gene expression in response to hormone signalling
43
Q

Provide examples of Group 2 Non-Steroid Nuclear Receptors and their ligands

A

T3R (Thyroid hormone) - Thyroid hormone
RAR (Retinoic Acid Receptor) - All-trans-retinoic acid
RXR (Retinoid X Receptor) - 9-cis-retinoic acid
VDR (Vitamin D Receptor) - Vitamin D
COUP-tf (Chicken Ovalbumin Upstream Promoter Transcription Factor) - Unknown ligand

44
Q

How do Group 2 Nuclear Receptors bind DNA as heterodimers?

A
  • Group 2 nuclear receptors bind a direct repeat sequence on DNA as heterodimers with RXR.
  • partner receptor (bound to its own ligand) forms a heterodimer with RXR, allowing for specific binding to DNA.
  • spacing and ligand type determine which partner receptor binds DNA in addition to RXR
45
Q

What is a Leucine zipper motif, and how do transcription factors with this motif dimerize?

A
  • Leucine zipper motifs = found in TFs that dimerize using leucine residues
  • Leucine residues every 7th amino acid interact & dimerize with other molecules of the same/different TFs.
  • Examples of transcription factors with leucine zipper motifs include CREB, C/EBP, Jun, Fos, and c-Myc
46
Q

How do Leucine zipper factors bind to DNA?

A
  • Leucine zipper factors DONT bind DNA directly; they require another leucine zipper factor for DNA binding.
  • Basic AAs near the leucine zipper motif (+) charged & can bind to (-) charged DNA
  • Dimerization occurs via hydrophobic interactions between leucine residues on the two leucine zipper factors
47
Q

What is the Helix-loop-helix motif, and how do Basic HLH factors bind to DNA?

A
  • HLH motif is a dimerization domain found in many TFs
  • Basic HLH factors bind to DNA by forming heterodimers with ubiquitous factors (e.g., E12) through their HLH domains.
  • DNA binding of basic HLH factors prevented by non-basic HLH proteins like Id (which lack a basic DNA-binding region)
48
Q

Describe the activation and repression domains in transcription factors

A
  • ADs & RDs lack distinct structural motifs
  • ADs more common than RDs and can be acidic, glutamine-rich, or proline-rich.
  • ADs often interact with the basal transcription apparatus to enhance binding & conformation of general TFs.
  • Some repressors act indirectly by blocking activator binding/interaction, others act directly by interacting with the basal transcription apparatus.
49
Q

How can transcription factors modify chromatin structure and influence gene expression?

A
  • some TFS have histone acetylase (HAT) or histone deacetylase (HDAC) activity, modifying histone proteins
  • HAT activity acetylates histones, loosening their binding to DNA and making it more accessible to TFs
  • HDAC activity removes acetyl groups, leading to compacted histone-DNA complexes.
  • Pioneer transcription factors like GATA4 and FoxA1 can bind to DNA even in condensed chromatin, opening it up for other factors
50
Q

How can tissue-specific or inducible gene expression be achieved through transcription factor regulation?

A
  • Tissue-specific synthesis of TFs → in specific gene expression patterns.
  • Regulation of splicing can generate different protein isoforms by alternatively splicing pre-mRNA.
  • Covalent modification such as phosphorylation can alter TF conformation and function.
  • Ligand binding to steroid receptors can release them from cytoplasm, allowing them to enter nucleus.
  • Interaction between factors like dimerization & recruitment of co-activators or co-repressors can influence gene expression

5BBG0205 Molecular Basis of Gene Expression (9 decks)

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