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MCB2020F > Eukaryotic Gene Regulation > Flashcards

Eukaryotic Gene Regulation Flashcards

1
Q

Eukaryotic vs Prokaryotic Gene Regulation

A

Eukaryotes have much greater levels of complexity for controlling of gene expression:
o Eukaryotic genomes are larger than prokaryotic genomes
o Chromatin structure in eukaryotes makes DNA unavailable to transcription machinery
o Additional RNA processing events occur in eukaryotes (Differential splicing)
o In eukaryotes, transcription takes place in the nucleus and translation takes place in the cytoplasm

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2
Q

What are Exons?

A

Sequences that end up in mature mRNA and reach the cytoplasm. However not all of them are translated.

Can be coding or non-coding.

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3
Q

What are Introns?

A

Segments of the gene that are in the primary transcript but are not included in the mRNA, they are removed during splicing

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4
Q

What is the cap site?

A

The beginning of the first exon

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5
Q

What is the Poly A site?

A

The end of the last exon (a sequence that causes synthesis of multiple Poly A sequences at the end of mRNA

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6
Q

Downstream Definition

A

3’-direction

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7
Q

upstream Definition

A

5’-Direction

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8
Q

TATA Box

A
  • ±30bp (in mammals) in the 5’ direction.

- It directs where RNA polymerase will assemble

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9
Q

Cis-acting elements

A

Promoters & Enhancers

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10
Q

Promoters

A
  • Usually directly adjacent to the gene (Usually on the 5’ flank)
  • Include transcription initiation site
  • Often have a TATA box
  • Allows basal level of transcription
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11
Q

Enhancers

A
  • Can be far away from the gene

- Augment or repress the basal level of transcription

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12
Q

Differences Promoters vs Enhancers

A
  • Both contain binding sites for regulatory transcription factors
  • Difference is their position in the genome
  • Proximal-Promoter is found from -1000 to -100bp upstream of TSS
  • Enhancers can be kilo bases up or downstream of TSS, or even within the gene itself
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13
Q

Levels of regulation in Eukaryotes

A
  1. Initiation of transcription
  2. Transcript processing (splicing, polyadenylation, stability)
  3. Export from nucleus
  4. Translation
  5. Modification/localisation of protein products.
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14
Q

Type of RNA Polymerases in Eukaryotes

A
  1. RNA Pol 1 - transcribes rRNA genes
  2. RNA Pol II - transcribes all protein-coding genes (mRNAs) and micro-RNAs
  3. RNA Pol III - transcribes tRNA genes and some small regulatory RNAs
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15
Q

Processing of RNA Pol II transcripts

A
  • Most RNA Pol II transcripts undergo further processing to generate mature mRNA
  • RNA splicing - removes introns
  • Addition of 5’ GTP cap - protects RNA from degradation
  • Cleavage of 3’ end and addition of 3’ PolyA tail
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16
Q

Basal Factors Definition

A

Basal transcription factors assist the binding of RNA pol II to promoters

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17
Q

Key components of Basal Factor Complex

A
  • TATA-Box binding protein (TBP): Is the first of several proteins to assemble at promoter and it binds to TATA box
  • TBP-associated factors (Transcription Ancillary Factors or ‘TAFs’): Binds to TBP assembled at TATA box
  • RNA Pol II associates with basal complex to form the Transcription Initiation Complex (TIC) and initiates basal level of transcription
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18
Q

Steps in Basal factor binding to core promoters

A

(Happens in all protein encoding genes)

  1. TBP binds to TATA box
  2. TAFs (transcription ancillary factors) bind to TBP
  3. RNA Pol II binds to TAFs
  4. Together the complex is referred to as the transcription initiation complex (TIC)
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19
Q

Transcription Factors Definition

A

Proteins that bind to DNA sequences within the proximal promoter or an enhancer to control the rate of transcription of a gene

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20
Q

Transcription Factors Function

A

Facilitate expression of genes in specific tissues at certain times or in response to specific condition

  • Can be tissue specific
  • complement can vary over time and space
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21
Q

General Method of transcription regulation by Transcription Factors

A
  • Interactions with components of the TIC
  • Modifying chromatin structure to make the transcription start site (TSS) more or less accessible
  • Regulatory TFs can be activators or repressors of transcription depending on the cell they are in (some are both).
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22
Q

Activators

A
  • TFs that increase rate of transcription
  • Either promote binding of TIC components/stabilising the TIC complex
  • OR recruiting co-activators (proteins that open chromatin and allow transcription)
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23
Q

Repressors

A
  • TFs that decrease rate of transcription
  • They can recruit co-repressors that directly prevent RNA Pol II from binding to the promoter
  • Can also recruit co-repressors that close chromatin structure
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24
Q

How do Transcription Factors bound to enhancers kb away from TSS interact with the TIC?

A

DNA is looped to a mediator complex

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25
Q

Transcription Factor Domains

A
  • DNA-binding domain (Of activator Proteins): facilitates binding to specific DNA sequences
    o Have alpha helices that interact with the major groove of DNA
    o Specific amino acids have high-affinity binding to specific nucleotide sequences
    o Best characterised motifs: Helix-Loop-Helix; Helix-Turn-helix; Zinc finger
  • Activation Domain OR Repression domain: interacts with TIC components or co-activators/repressors
  • Some have ligand-binding domains (e.g. steroid-receptors)
  • Dimerization domains: specialised for polypeptide-polypeptide interactions. 2 amino-acid monomers come together forms a dimer which becomes the DNA binding region
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26
Q

Leucine Zipper

A
  • Common example of a TF with a dimerisation domain
  • Amino acid sequence forms alpha helices with leucines protruding seven base pairs.
  • These leucines then interact with each other in a protein-protein interaction resulting in the 2 monomers coming together and forming a dimer via the hydrophobic interactions of the leucines, all orientated on one side of an alpha helix.
  • The dimerization of two monomers then constitutes or forms the DNA binding domain of the dimer.
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27
Q

Why does dimerisation increase capacity for control of gene expression?

A
  • You can mix and match different monomers to form different dimers, homodimers, and heterodimers, that have a different DNA binding specificity.
  • So by using fewer proteins, you can generate more different transcription factors consisting of dimers that can then regulate gene expression and have different DNA specificities
  • e.g. Jun-Jun (homodimer) binds to the same enhancer sequence as Jun-For, but has different affinities
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28
Q

Levels of control of Transcription Factor activity

A
  • Allosteric interactions with small molecules
  • Post-translational modification to TFs
  • Transcription factor cascades (TFs regulating expression of other TFs)
  • DNA methylation: Occurs in CpG islands (region of lots of C-G nucleotide repeats) and can block TF binding in 2 ways:
    o Alters patterns of H-bonding in major groove
    o Recruits chromatin remodelling proteins leading to formation of “repressive” chromatin
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29
Q

Integration of cellular information

A
  • One gene can be regulated by many transcription factors
  • One TF can regulate many genes
  • There are also many co-activators/co-repressors in a cell
  • This allows for Combinational Regulation
  • This complexity allows for differentiation of cell types and response to external stimuli.
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30
Q

Reporter Assays Definition

A

Linking of coding regions of reporter gene to promoter region in a piece of synthetic DNA (Like a plasmid)

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31
Q

Reporter Assays Function

A

Can be used to investigate the role of specific transcription factors in transcriptional regulation

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32
Q

in vivo Reporter Assays

A

in vivo = in a whole organisms

- Reporter genes stably integrated into transgenic organisms (like seeds)

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33
Q

in vitro Reporter Assays

A

in vitro = in the lab in a culture

  • Reporter genes transfected into cells in culture
  • Can be stable of transient transfection
34
Q

Chromatin Structure

A
  • Dynamic and regulates transcription in eukaryotes
  • DNA complexed by histone proteins into nucleosomes
  • Nucleosomes condense together to form chromatin
  • Chromatin reduces transcription by reducing binding of DNA to basal factors and RNA pol II to very low levels
  • Chromatin has localized regions of active chromatin (i.e. can be compacted and uncompacted at different points on same chromatin)
35
Q

Chromatin regulation of transcription

A
  • Nucleosomes can sequester at or be positioned at promoters/enhancers and make them inaccessible to RNA Pol II and transcription factors
  • Histone modification can change how tightly the DNA is associated with nucleosomes
  • Chromatin remodeling or moving of nucleosomes can occur (DNA methylation can cause chromatin remodeling)
36
Q

Histone Modification

A

N-terminal tails of histones can be modified:

  • Can be through methylation, acetylation, phosphorylation, ubiquitination
  • Can affect nucleosome interaction with other nucleosomes and with regulatory proteins
  • Can affect higher-order chromatin structure

TFs can recruit co-repressors with histone deacetylation (HDAC) activity

37
Q

Lysine acetylation

A
  • Form of Histone Modification
  • Causes loss of positive charge on histone tail which decreases its interactions with DNA
  • Positive charge is lost on side chain amino group
  • Decreases electrostatic interaction with DNA phosphate group of the backbone
  • Done by co-activators with histone acetylation (HAT) activity (recruited by TFs)
38
Q

Chromatin Remodelling

A
  • Nucleosomes can be repositioned or removed by chromatin remodelling complexes:
    • After remodelling DNA at promoters/enhancers they become more accessible to transcription factors
    • Can be assayed using DNase digestion
  • ATP dependant process (nucleosome needs to be MOVED)
  • Uses proteins histone acetyl transferases to open up chromatin, and histone deacytl transferases to close it
39
Q

Levels of Post-Transcriptional regulation of gene expression

A
  1. mRNA level
  2. Translational regulation
  3. Protein regulation (post-translational regulation)
40
Q

Splicing

A
  • The splicing of primary transcripts into mRNAs that code different proteins
  • Alternative splicing = different versions of proteins being coded by mixing and matching combinations of exons
  • Includes the removal of introns
  • Eukaryotic genes contain many exons which enables production of different mRNAs by cell-specific alternative splicing during RNA processing
41
Q

The Spliceosome

A
  • Massive macromolecular complex containing 5 small nuclear ribonucleoproteins (snRNPs) and up to 300 other proteins
  • snRNPs are called U1, U2, U4, U5 & U6
  • Each contains a single short RNA molecule
42
Q

siRNA

A
  • Specialized, small, double stranded RNAs that prevent expression of specific genes through complementary base pairing
  • Synthetic or exogenous (e.g. from a virus)
  • Called small-interfering (si) RNA
  • Small (12-30 nt)
  • Both siRNA and miRNA can inhibit gene expression by post-transcriptional mechanisms
43
Q

miRNA

A
  • Endogenous genome-encoding molecule
  • Specialized, small (12-30nt), double stranded RNAs that prevent expression of specific genes through complementary base pairing
  • First miRNAs identified in C. elegans
  • Both siRNA and miRNA can inhibit gene expression by posttranscriptional mechanisms
  • Most are transcribed by RNA polymerase II from noncoding DNA regions that generate short dsRNA hairpins.
  • About 25% from introns of protein-coding transcripts and about 75% from transcripts without open reading frames.
44
Q

miRNA Processing

A
  • Drosha: a specialsed RNase that excises the stem-loop from primary miRNA to generate a pre-mRNA
  • Dicer: a specialsed RNase then processes pre-miRNA to a mature duplex miRNA
  • 1 strand is incorporated into miRNA-induced silencing complex (RISC) which is a large polypeptide RNase complex.
45
Q

miRNA & siRNA down-regulation of gene expression

A
  1. When complementarity is perfect: Target mRNA is degraded

2. When complementarity is imperfect: translation of mRNA target is repressed

46
Q

RNA editing

A
  • Changing of nucleotides results in different codon which leads to different protein coding
  • E.g. Reaction catalyzed by cytosine deaminase converts cytosine to uracil
47
Q

RNA Stability

A
  • Rate of turnover depends on rate of synthesis and degradation
  • Longer life of mRNA = more protein that can be produced
    o Cap and poly A tail offer protection against RNases (longer poly a tail = longer half life)
    o If removed the stability of the mRNA is reduced
    o Regulatory elements (eg secondary structure) in UTRs can promote mRNA degradation
48
Q

Translational Regulation

A
  • Regulation can take place at any step
    • Assembly of initiation complex (including elongation factors)
    • Large subunit binds
    • Translation begins
    • Elongation of peptide chain occurs
  • NOT ALL mRNAs are translated immediately
    o Some are stored, e.g. oocytes (egg cells) translation only occurs after fertilization
49
Q

Post-translational Regulation

A
  • Protein turnover and degradation
  • Acetylation of histones
  • Glycosylation of cell surface proteins
  • Cascades of phosphorylation and dephosphorylation function in transmission of cell signals from membrane to nucleus
50
Q

Ubiquitination

A

Covalent attachment of ubiquitin to protein results in these proteins becoming targets for degradation by the proteosome

51
Q

Methods of Cell Signalling

A
  • Endocrine signalling:
    • Hormones travel to distant targets and bind to receptors in/on target cells
  • Paracrine signalling:
    • Secretory cells releases hormones which targets adjacent cells
  • Autocrine signalling:
    • Receptor for signalling molecule is on the same cell
  • Plasma-membrane attached proteins:
    • Signal is the attachment of proteins to adjacent cells
52
Q

Types of hormones & their functions

A
  1. Steroid hormones:
    • Regulate metabolism, salt & water balance, inflammatory responses, sexual function
  2. Amino Acid derivates:
    • Regulate smooth muscle function, blood pressure, cardiac rate
  3. Peptide hormones:
    • Regulate processes in all tissues, including release of other hormones

(4. Protein/glycoprotein hormones)

53
Q

Signalling cascade for intracellular receptors

A
  • Steroid Receptors
  • Hormone is usually hydrophobic (receptor is inside of cell) so can pass into cell without a channel
  • In absence of hormone, receptor is inactive due to the presence of inhibitor proteins on LBD (ligand-binding domain)
  • Binding of hormone to LBD causes conformational change in GR (glucocorticoid receptor)
    o Leads to release of inhibiting proteins
    o Leads to dimerization of receptor which increases its affinity for DNA
  • Allows receptor to be translocated into nucleus, they regulate rate of transcription and transcription initiation
  • GR recognizes specific sequences/promoters and bind to DNA promoter regions as dimers
    o Regulate transcription via recruitment of co-activators
  • Response elements or cis-elements are palindromic DNA sites that bind several major nuclear receptors as dimers
    o Nuclear receptors bind to DNA with high affinity and specificity
54
Q

Domains of an intracellular receptor

A
  • Activation Domain
  • DNA-binding domain (zinc fingers)
  • Ligand-Bind Domain
55
Q

Allosteric effect of steroid-receptor binding

A

Steroid hormone binding to receptor protein causes allosteric changes in the receptor protein that greatly increases the affinity of the DNA binding domain for the enhancer region of the DNA

56
Q

Cell Surface Receptors Signalling cascade

A
  1. Hormone released from signalling cell
  2. Hormone travels various distances to target cells through blood; recognises the target cell by the protein receptor on the membrane
  3. Binding of hormone to cell surface receptor will activate the receptor, usually causing a conformational change = transduction of signal into cell
  4. Signal transduction proteins and 2nd messengers target activation of effector protein (enzymes, transcription factors etc.)
  5. This causes the desired response (modification of cellular metabolism, function, movement etc.)
  6. Transcription factors (if they are the effector protein) can move into the nucleus and affect modification of gene expression/developement.
57
Q

What are the 3 cell surface receptor Superfamilies?

A
  • Classified as Superfamily if there is a conserved OVERALL structure
    1. G-protein-coupled receptors: integral membrane proteins with intracellular site for a GTP-binding protein
    2. Single-transmembrane-segment catalytic receptors (Tyrosine kinase or guanylyl cyclase): large intracellular domain with enzyme domain inside the cell
    3. Oligomeric ion channels: bind ligands and in response open or close the channel through the membrane (Ligand-gated channels)
58
Q

What is a GPCR

A

G-Protein Coupled Receptor

59
Q

What are the key players in a GPCR signalling cascade?

A
  1. G-Protein Cell Receptor (GPCR)
  2. G-Protein
  3. Effector enzyme (adenylyl cyclase)
  4. Second messenger (cAMP)
  5. Kinase - protein kinase A (PKA)
    • Contains 2 catalytic subunits (C) and 2 regulatory subunits (R)
  6. Transcription factors; cAMP-response element binding proteins (CREB)
60
Q

GPCR Action Cascade

A
  1. Ligand binds to GPCR which activates G-protein
  2. G-protein targets adenylyl cyclase (membrane protein) which synthesizes cAMP from ATP
  3. Quick changes in conc of second messenger cAMP actives kinase PKA
  4. Activation of PKA (by cAMP binding to regularity subunits) frees catalytic subunits which travel into the nucleus
  5. They phosphorylate transcription factors to activate them.
    a. PKA phosphorylates CREB which can then bind to its response element on DNA
    b. Recruits coactivator and increases rate of transcription initiation
61
Q

Features of a GPCR

A
  • GPRs are 7 Transmembrane-segment (TMS) Integral membrane proteins
  • Binding of hormone to GPCR induces a conformation change that activates a trimeric GTP-binding protein, also known as G protein
  • Activated G protein triggers various cellular effects:
    o Activation of adenylyl and guanylyl cyclases
    o Activation of phospholipases
    o Activation of ion channels
    o Many end in transcription by activating kinases which activate transcription factors
  • Transmembrane segments are alpha helices
    o Amphipathic alpha helix’s
    o Amino terminus is usually extra-cellular
    o C-terminal protrudes into interior of cell
62
Q

What is the GPCR for vision?

A

Rhodopsin

63
Q

Examples of Enzyme-linked receptors

A

EGF (Epidermal growth factor) Receptor
Insulin Receptor
Natriuretic peptide receptor

64
Q

RTK full name and examples

A

RTK = Receptor Tyrosine Kinase

Examples: EGF receptor & Insulin Receptor

65
Q

Features of Enzymes-linked receptors

A
  • Extracellular domain and a single alpha-helix per protein which spans the width of the membrane
  • Intracellular tyrosine kinase domains which becomes enzymatically activated once receptor binds to ligand -> phosphorylation of tyrosine residues (including itself, autophosphorylation)
66
Q

Features of RTKs

A
  • Ligand binding induces conformational change and dimerisation
  • EGF Receptor:
    • Receptor monomers aren’t associated in absence of ligand.
    • Ligand binding induces binding of 2 monomers to form a dimer which reconstitutes intracellular tyrosine kinase domain
  • Insulin Receptor:
    • 2 monomers are already associated with sulphide bonds before ligand binding,
    • ligand binding causes conformational change resulting in a more tightly associated dimer which forms an enzymatically active tyrosine kinase domain
67
Q

Ras & Mitogen Activated Protein Kinase (MAPK) cascade

A
  • Ligand binds to RTK causing dimerization which activates the tyrosine kinase domain
  • Results in autophosphorylation = 1 monomer phosphorylates the other and vice versa forming phosphorylated tyrosine residues
    o Phosphorylated tyrosine residues function in recruiting adapter proteins.
    o Forms a complex of adapter proteins (first one is GRB2)
    o Second adapter protein is SOS which helps activate Ras
  • SOS activates G-Protein called Ras (GDP dissociates & GTP associates) which activates Raf (a MAP kinase kinase kinase)
    o Ras is anchored in membrane by lipid residue
  • Raf activates MAPK pathways
  • 3-tiered kinase signaling pathways
    o MAP kinase kinase kinase phosphorylates MAP kinase kinase which in turn phosphorylates MAP kinase
  • MAPK phosphorylates downstream target proteins
    o Often transcription factors in nucleus in order to affect gene transcription and therefore protein synthesis
    o Or cytosolic targets (promoting growth and division), in order to effect a cellular response
68
Q

What is a phosphorelay system?

A

The successive phosphorylation of kinases

69
Q

Function of phosphorelay systems

A

Phosphorylation by MAPKs acts as a switch to turn on or turn off the activity of the substrate proteins

70
Q

2 Types of G-Proteins

A
  • Heterotrimeric G proteins (3 subunits; alpha, beta, gama)

- small G proteins (1 subunits)

71
Q

Do G-Proteins phosphorylate targets?

A

No (Kinases phosphorylate targets), rather they cause conformational changes at targets

72
Q

Heterotrimeric G-Protein mechanism of action

A
  1. Binding of hormone induces a conformational change in receptor
  2. Activated receptor binds to G∝ subunit
  3. Activated receptor causes conformational change in G∝, triggering dissociation of GDP
  4. Binding of GTP to G∝ triggers dissociation of G∝ both from the receptor and from G(βγ)
  5. Hormone dissociates from receptor, G∝ binds to effector, activating it
  6. Hydrolysis of GTP to GDP causes G∝ to dissociate from effector and re-associate with G(βγ)
    • > Back at resting state
73
Q

What is a 2nd messenger?

A

A small molecule who’s concentration in the cell can be changed very quickly

74
Q

Function of a second messenger

A
  • Released when hormone binds to its extracellular receptor

- It then activates or inhibits processes in the cytosol or nucleus

75
Q

IP3/DAG mediated signal transduction pathway

A
  • Effector: Phospholipase C which cleaves second messengers from membrane itself
  • 2nd Messengers = DAG (Diacylglycerol) & IP3
    G-Protein = Gq-G protein
  1. DAG activates kinase (Protein Kinase C)
  2. PKC phosphorylates target proteins -> activation -> cellular response
  3. IP3 affects whether ion channels in ER are open or closed. Binds to them to open them which releases Ca2+ that was stored inside
  4. Ca2+ is a 2nd messenger which can activate Ca2+ dependant kinases (Does similar things to PKC but with different specificity)
76
Q

Kinases General Information

A
  • Kinases add phosphate groups to specific amino acids
  • Phosphatases remove them
  • Many proteins, including transcription factors, regulated by kinases
  • Serine phosphorylates to phosphoserine
  • Threonine phosphorylates to phosphothreonine
  • Tyrosine phosphorylates to phosphotyrosine
77
Q

Amplification

A
  • Signals are amplified during signal transduction of enzymes
  • cAMP & signal amplification (same principle applies to any series of kinases):
    o GPCR activated by binding of ligand -> activates G-protein -> activates adenylyl cyclase
    o Each activated AC generates many cAMP mols (1 hormone activates 1 G-protein can activate thousands of cAMP becayse of enzymatic activity of AC)
    o cAMP mols stim PKA; each PKA phosphorylates many kinases
    o Each kinase phosphorylates many targets (including kinases)
78
Q

Importance of switching off signals

A
  • Failure to switch off G-proteins leads to disease

- Can result in over production/stimulation of a cellular response

79
Q

Result of the cholera toxin

A
  • Irreversibly modifies G-protein causing permanent activation of G-protein
  • It ADP-ribosinates target proteins (requires NAD+)
    o This means it adds an ADP-ribose residue on the G∝ subunit
  • G∝ is conformationally changed which inhibits GTPase activity of G∝ subunit -> GTP cannot be hydrolyzed to GDP (i.e. it can’t be switched off)
  • This leads to the persistent activation of adenylyl cyclase.
  • Leads to massive production of cAMP
  • Fluid secretion in intestine is regulated by GPCR cAMP pathway
    o Result of cholera toxin is massive fluid release from intestines and diarrhea (death can follow shortly from dehydration)
80
Q

Ras oncogene Effect

A
  • Normal RAS is inactive until it becomes activated by binding of growth factors to their receptors
  • Oncogenic forms (mutated forms that lead to cancer) of RAS are constantly activated -> unregulated cell proliferation even without growth factor
    o This often forms tumors and can lead to cancer
81
Q

GPCR Desensitisation

A
  • In addition to the heterotrimeric G proteins, GPCRs also bind 2 other classes of mols
    o Family of protein kinases known as GPCR kinases (GRKs) {phosphorylate the receptor itself}
    o Family of adapter and scaffolding proteins known as beta-arrestins
  • These 2 families work together to desensitize the GPCRs, “arresting” the G-protein activation of GPCRs
82
Q

Inhibition of Rhodopsin signalling

A

o Rhodopsin desensitized to the amount of light

o After rhodopsin exposed to continues amounts of bright light, it becomes less sensitive to light because its C-terminal tale becomes phosphorylated by Rhodopsin kinase

o Once phosphorylated to a certain extent then protein Arrestin binds and Rhodopsin is fully switched off.

MCB2020F (6 decks)

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