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Genes and Genomics > Transcriptional Control > Flashcards

Transcriptional Control Flashcards

1
Q

Eukaryotic RNAP

A
  • 3 kinds
    RNAPI: rRNA (nucleolus)
    RNAPII: mRNA
    RNAPIII: tRNA, small RNA
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2
Q

Bacterial vs. Eukaryotic RNAPs

A
  • structural ortholog elements
  • however, eukaryotic RNAP cannot bind in a sequence specific way on their own
  • require transcription factors to recognise promoter and form bubble
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3
Q

Eukaryotic Basal Transcription

A
  • need to initate transcript
  • assembled initation complex identical for every RNAPII gene giving low levels of unregulated transcription
  • further gene specific TF give regulated levels of expressoin
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4
Q

Eukaryotic Promoter Model

A
  • proximal gene specific TF

- distal gene specific TF (loop around to interact with start site basal initiation complex)

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5
Q

TATA Box

A
  • 25/-30 upstream from gene
  • recognised by TFIID
  • stronger binding with higher sequence specificity
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6
Q

TFIID

A
  • multiprotein complex of TATA binding protein and other subunits
  • binds sequence specifically to TATA box
  • binding stabilised by TFIIA/TFIIB
  • TATA binding protein kinks DNA
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7
Q

Electrophoretic Mobility Shift Assay

A
  • shows how TFIIB stabilises binding
  • with presence of TFIIB you get strong binding of TBP to the DNA
  • bending DNA is energetically inefficient
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8
Q

Other Promoter Elements

A
  • initiator element (+1)

- downstream promoter element (+28/+34)

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9
Q

Additional Basal Factor

A
  • TFIIF binds RNAPII in solutoin and delivers it to TFIID/B/DNA on the promoter
  • TFIIE and TFIIH responsible for 3 critical functions in transcription: phosphorylation of RNAPII, promoter melting, promoter clearance
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10
Q

Phosphorylation of RNAPII

A
  • C terminal tail of 26 repeats of a 7 residue sequence phosphorylated by TFIIH kinase
  • Hypophosphorylation: CTD associated with intitiation complex
  • Hyperphosphorylation: CTD associated with elongation competent RNAPII
  • regulates 5’ capping, assembly of spliceosomes, binding of cleavage/polyadenylation complex
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11
Q

Promoter Melting

A
  • TFIIH melts dsDNA around start site to form bubble
  • bubble unstable and if initiation doesn’t occur in this time span ATP hydrolysis is needed
  • regulates rate of initiation of transcription
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12
Q

Regulatory Regions

A
  • in addition to promoters, eukaryotic genes contain binding sites for gene specific TFs
  • sequence specific binding of gene specific TFs allows genes to achieve and maintain controlled levels of tissue specific expression patterns
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13
Q

Gene Specific TF

A
  • eukaryotic factors usually transcriptional activators
  • bind to subset of genes through sequence specific DBD
  • modulate activity of promoter bound transcriptional machinery through activation domains
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14
Q

DNA Footprinting

A
  • use DNaseI to cleave sequence of DNA bound with and without protein
  • with protein will show gaps in blotting assay where nuclease cannot cut protein bound DNA
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15
Q

DNA - Protein Interactions

A
  • electrostatic bonds (long range)
  • hydrogen bonds
  • VDW forces
  • hydrophobic interactions
  • structural complementarity
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16
Q

Sequence Specific Interactions to DNA

A
  • electrostatic interactions provide stabilising energy but not specificity
  • TF binding doesn’t cause unwinding
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17
Q

B DNA

A
  • antiparallel right hand double helix
  • minor and major groove
  • bases project into interior
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18
Q

Groove Contacts

A
  • absolute recognition of the four different base pairs is only possible via the major groove
  • asymmetric hydrogen donor acceptors (G:C)
  • bulk methyl group on T residues
  • minor groove binding can only distinguish A:T from G:C (not flipping of each pairing)
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19
Q

Helix Turn Helix

A
  • two alpha helices separated by a loop
  • recognition helix (major groove), N terminal helix
  • side chains of recognition helix hake base specific contacts
  • form dimers to recognise palindromic seuqneces
20
Q

Leucine Zipper

A
  • cJUN/cFOS heterodimer
  • encoded by separate genes (oncogenes)
  • bind to form Y shaped DBD
  • binding of helices to form Y shape DBD involves hydrophobic interactions between regularly spaced leucines on both helices
  • forms a tight leucine zipper
21
Q

Helix Loop Helix

A
  • cMAX can form homodimer or heterdimer with cMYC
  • complex version of leucine zipper containing loop interrupting a helixes
  • binding of helices to for Y shape still involves hydrophobic interactions between regularly spaced leucines on both helices
22
Q

Zinc Finger Domains

A
  • zinc atom coordinated by cysteine or histidine
  • no bacterial
  • B turn and a helix held together by coordinated ZN atom (4 residues)
  • work in tandem with each finger recognising a 3 nucleotide target site in DNA
23
Q

p53 DBD

A
  • tumor suppressor causing apoptosis
  • central domain binding DNA in a sequence specific manner
  • no structural similarity to any other known DBD
24
Q

Activation Domains

A
  • stimulate activity of basal transcriptional machinery
  • unusual amino acid composition characteristic of IDPs
  • no defined 3D structure: high charge density and prolines prevent this
  • versatile binding to many targets
  • aid recruitment of basal factors, create open chromatin, stimulate enzymatic activities
25
Q

Partial Nuclease Digestion

A
  • micrococcal nuclease digest linker DNA between nucleosomes

- generation of distinct DNA fragments containing one or more repeat units

26
Q

Nucleosomes

A
  • 4 core histones: H2A, H2B, H3, H4

- all four histones contain central histone fold (dimerization motif)

27
Q

Histone Dimerization

A
  • tight binding with electrostatic and hydrophobic forces
28
Q

Histone Octamer Assembly

A
  • 2 copies each of H4/H3 form H3-H4 tetramer
  • H2A-H2B dimer forms
  • two components brought together to form the histone octamer
29
Q

Histone Evolution

A
  • highly conserved in eukaryotes
  • probably emerged early during evolution
  • likely they evolved to stabilize and protect DNA in extreme conditions of euryarchaeota
30
Q

Nucleosome DNA Interactions

A
  • rigid DNA bent around nucleosome unfavourably in two tight circles due to interaction strength
    1. ionic/H bonds (histone positive)
    2. non polar contact
    3. intercalation of arginines to phosphates across the groove
    4. amide phosphate bind interactions
    5. histone helix dipoles
31
Q

DNA Distortion

A
  • major groove widened to prevent TF binding

- recudes spatial complementarity

32
Q

Linker Histones

A
  • H1/H5 stabilise interactions between nucleosomes in compacted chromatin
  • organise entry and exit ponits of DNA
33
Q

Chromatin Organisation

A
  • DNA wrapped around histone octamer as nucleosome
  • nucleosomes held together by H1 linker protein
  • forms chromatine fibers
34
Q

Solenoid

A
  • The solenoid structure of chromatin is a model for the structure of the 30 nm fibre. It is a secondary chromatin structure which helps to package eukaryotic DNA into the nucleus.
  • can be open or compact
35
Q

Disproving Solenoid Model

A
  • doubt about validity of model in vivo
  • chromEMT saw irregular patterns of DNA and no evidence of solenoids
  • still have looser and tighter conformations
  • concluded that nucleosomes assemble into disordered chains with different arrangements
  • little evidence for regular helical structure
  • chains are flexible and bend at various length to achieve levels of compaction
36
Q

Histone N Termini

A
  • N termini sequences control gene regulatory properties
  • highly flexible
  • emerge from nucleosome
  • acetylated by conserved lysines
  • compact to interact with backbone
37
Q

Histone Acetylation

A
  • acetylation of N termini creates open chromatin
  • exposed DNA closer to feed into RNAP from nucleosome
  • opens dna
  • deacetylation allows positively charged N termini to bind DNA on neighbouring nucleosomes
38
Q

Chromatin Opening

A
  • gene specific TFs recognise target sequence in compacted chromatin
  • recruit other enzymes
  • localised opening/closing
39
Q

Methylation

A
  • effect depends on position/residue on histone

- nucleosomes in heterochromatin usually methylated

40
Q

c-Myc

A
  • proto oncoprotein
  • 60-70% of cancer dependent on mutation
  • is a TF
41
Q

c Myc Function

A
  • regulates expression of genes involved in cell proliferation
  • increases metabolic rate
  • increased nutrient uptake, enhanced glycolysis, etc
42
Q

c Myc Overexpression

A
  • normal WT protein but translocation to another chromosome under the control of active immunoglobin H promoter
43
Q

c Myc stability

A
  • short WT half life
  • stability of cMyc increased between due to the presence of mutations in particular regions of the proteins that initiate its degradation
  • prevent degradation supporting growth of cancers
44
Q

c Myc DBD

A
  • helix loop helix motif
  • associates with cMax with HLH DBD
  • positively charged arginine residues contact negatively charged phosphates on DNA
  • hydrophobic leucine form leucine zipper
45
Q

c Myc Structure

A
  • intrinsically disordered aside from HLH region
  • transcriptional activation domain
  • N termal IDP can interact with large number of TF in specific by conformationally flexible manner
  • conserved residues tend to be hydrophobic and bulky
  • islands of low homology
  • transient secondary structure and modelling with molecular dynamics simulations

Genes and Genomics (13 decks)

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