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genes + molecules > gene expression + chromatin structure > Flashcards

gene expression + chromatin structure Flashcards

1
Q

why do we regulate gene expression?

A

so different cells can express different genes and perform different functions
differentiation depends on gene expression changes

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2
Q

what are the 5 ways in which we can regulate gene expression? …… TBC?

A
  1. transcriptional control
  2. RNA processing control and mRNA degradation control
  3. RNA transport control
  4. translational control
  5. chromatin structure (eukaryotes)
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3
Q

why does it make the most sense to regulate transcription to regulate gene expression?

A

it is the top of the regulatory hierarchy
you don’t make mRNA unless you’re going to make protein - a waste of energy if you regulate after this point
reduces superfluous intermediates

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4
Q

how is the eukaryotic gene organised?

A

has a regulatory and transcribed region, with the coding region (that is translated) appearing within the transcribed region

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5
Q

what is contained in the regulatory region of the eukaryotic gene?

A

enhancers + promoters (TATA box)

simple switches + complicated micropressors

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6
Q

what is contained in the transcribed region of the eukaryotic gene?

A

transcription start site (TSS)
5’ UTR
the coding region:
[exons
introns
the stop codon for translation]
3’ UTR
cleavage + polyA signal

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7
Q

what is the promoter?

A

the TATA box
where RNA polymerase makes contact with DNA

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8
Q

what do enhancers do?

A

regulate what is going on at the promoter

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9
Q

what are transcription factors?

A

proteins that bind to regulatory DNA sequences at specific recognition sequences + distort DNA structure

they make it easier/harder for the RNA Pol complex to bind + transcribe RNA

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10
Q

what does ‘reading’ a DNA sequence mean?

A

DNA binding proteins have a 3D architecture that recognises the specific 3D structure of the the outside of the DNA strand

DNA bps form specific interactions with nucleotides on major groove of B-DNA strand

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11
Q

what are the key DNA binding protein domains?

A

helix-turn-helix motif
zinc finger
leucine zipper

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12
Q

what is a helix-turn-helix motif?

A

2 helices held at a fixed angle
COO- helix = recognition helix: fits into the major groove of DNA
amino acid side chains recognise specific DNA binding sequences
other helix = residues contact the DNA backbone

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13
Q

what is a zinc finger?

A
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14
Q

what is a leucine zipper?

A
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15
Q

how do we measure DNA-protein interactions to ensure DNA binding proteins have bound?

A

electrophoretic mobility shift assay (EMSA)
gel electrophoresis followed by autoradiography

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16
Q

why are genes switched on/off in prokaryotic organisms?

A

its how bacteria adapt to the changes in composition of their growth medium

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17
Q

what is an example of a repressor in bacteria?

A

tryptophan repressor
(h-t-h motif)
used when tryptophan is available in the medium as there is then no point in making it in the cell

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18
Q

how is the code for making tryptophan in E coli encoded?

A

trp operon is a polycistronic mRNA molecule

coordinately controlled + on unless repressed

5 genes are needed to produce 3 enzymes for the 3 step synthesis

5 genes close together on bacterial chromosome, under control of 1 promoter that produces one massive mRNA that produces all 5 enzymes when translated

operator sits between promoter and 5 genes: controls whether RNA polymerase can bind or not

gene is normally on but tryptophan repressor can block access : genes not transcribed + enzymes not produced on

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19
Q

what gene controls production of tryp repressor?

A

trpR

tryp repressor produced frequently in inactive state, activated by tryptophan itself binding (indicating it is present in high enough levels int he cell) – repressor activation is concentration-dependent

activated tryp repressor can bind to operator to inhibit further production of tryptophan

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20
Q

what is an example of a gene that is typically off ?

A

lac - the lactose-processing gene - encoding beta-galactosidase

typically a repressor bound to the operator that controls lac genes

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21
Q

how do you activate the lac genes?

A

allolactose binds to lac repressor (that is bound to operator) and causes it to conformationally change and unbind from DNA, allowing for transcription + higher levels of lactose metabolism

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22
Q

what is negative gene regulation?

A

signalling molecules do not interact directly with DNA

they interact with repressor proteins

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23
Q

what is positive gene regulation?

A

a signalling molecule binds with an activator to generate a complex that interacts with DNA

directly stimulates gene expression by increasing affinity of RNA polymerase for the promoter

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24
Q

what is constitutive vs regulated expression?

A

C = expressed all the time
R = expressed under a particular set of physiological conditions

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25
Q

why does E. coli need to be able to metabolise lactose?

A

mainly rely on glucose as source of carbon and energy but can use lactose if this is scarce

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26
Q

what is the action of beta-galactosidase?

A

hydrolyses lactose into galactose and glucose

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27
Q

what 2 other proteins are synthesised together with beta-galactosidase? what does this tell us?

A

galactoside permease + thiogalactoside transacetylase

expression levels of a set of enzymes that all contribute to the adaptation to a given change in the environment, change together

coordinated unit = operon

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28
Q

what is the structure of a prokaryotic operon?

A

regulator gene : encodes regulatory (repressor) protein
operator site: regulatory DNA sequence
set of structural genes

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29
Q

what binds to the operator site in prokaryotes to prevent transcription of the structural genes?

A

the repressor protein coded for earlier in the operon

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30
Q

what is the structure of the lac repressor?

A

tetramer of 37kDa subunits

2 pairs of subunits come together to form the DNA binding unit

bound tightly to operator in absence of lactose

31
Q

how does the lac repressor block transcription?

A

binds to operator site which is adjacent to and downstream of promoter site, blocking the progress of RNA polymerase

32
Q

where does RNA polymerase bind to an operon?

A

promoter

33
Q

how does lac repressor locate operator site?

A

it binds 4 x10^6 times as strongly to the operator DNA as to random sites in the genome

rate constant is very high, repressor finds operator by diffusing along DNA molecule (1D search)

when 1 DNA-binding unit of lac repressor binds to operator site, the other unit can bind to 1 of a few sites with similar sequences and the DNA in between forms a loop

34
Q

what is allolactose?

A

galactose and glucose bound with an a-1,6 linkage

the inducer of the lac operon

a side product of the beta-galactosidase reaction

35
Q

how does the inducer allolactose work?

A

binds to lac repressor and greatly reduces repressor’s affinity for the operator

binding leads to 2 subtle conformational changes so 2 DNA binding domains can no longer contact DNA at same time

36
Q

what is a corepressor?

A

a small molecule that binds a repressor (such as pur), allowing it to bind DNA specifically, blocking transcription

37
Q

what is catabolite repression?

A

when the presence of an abundant energy source represses the production of enzymes used for metabolising other sugars

38
Q

what does glucose do to levels of cAMP in E. coli? what does it mean for other enzymes when glucose is removed?

A

it lowers it

allows transcription of other catabolic enzymes by acting through the identical-subunit-dimer: catabolite activator protein (CAP)

39
Q

where does CAP bind in the lac operon?

A

an inverted repeat at approx -61 from TSS

bends DNA in a manner favouring interactions with RNA pol

40
Q

what activates CAP?

A

cAMP

41
Q

how does CAP-cAMP complex increase rate of transcription?

A

creates energetically favourable contacts between CAP and RNA pol that increase likelihood of transcription being initiated at CAP-bound sites

increases 50x

42
Q

outline the mechanism of turning on and off the lac operon, depending on both mechanisms

A

requires there to be no glucose in the cell and also requires the presence of lactose

allolactose causes lac repressor to unbind from DNA

low glucose causes high cAMP -> CAP activated -> higher levels of transcription of lac genes

43
Q

what is a general transcription factor?

A

assembles the promoter machinery (basal transcriptional machinery)

44
Q

what is a regulatory transcription factor?

A

acts at the enhancer to slow down/speed up transcription

(activator protein)

45
Q

what is the key aim of promoter machinery assembly?

A

binding of TATA box binding protein (TBBP) to TATA box

46
Q

what is the mechanism of promoter machinery assembly?

A
  1. TBBP subunit of TFIID binds the TATA box
  2. TFIIB joins complex + assembles at promoter
  3. TFIIF brings RNA polymerase II into complex
  4. TFIIE and TFIIH join complex
  5. TFIIH has kinase activity and phosphorylates C terminal tail (CTD) of RNA polymerase II
  6. RNA polymerase II is released from tight binding of DNA so it can move along to gene to catalyse transcription

(all general TFs)

47
Q

what needs to happen after promoter machinery assembly?

A

regulatory TFs need to bind to enhancers to cause transcription

48
Q

where are enhancers and how do they work?

A

can be up to 25 kB up or downstream

reg TFs bind to them and the intervening DNA loops out so the TF and enhancer combo can directly communicate with the basal transcriptional machinery at the promoter

favour or discourage transcription

49
Q

what are the necessary things that have to occur for regulators to have their effect?

A

promoter bound to basal machinery

enhancer bound to TF

50
Q

what is the structure of a transcription factor?

A

TFs have modular structure:
a DNA binding domain (h-t-h)
activation/repression domain (acidic, glutamate/proline rich)

51
Q

which organisms tend to use enhancer sequences?

A

eukaryotes

52
Q

how does communication between basal machinery and enhancer activate the BTM?

A
53
Q

what is the structure of a nucleosome, assuming structure of a histone is known?

A

repeating units of histone octamers with stretches of linker DNA

supplementary 146 bp of DNA

shortens DNA from 680 to 100 angstroms

main structural units of chromatin

54
Q

how is DNA wrapped around histones?

A

200 bp of -ve DNA wraps histone proteins due to high conc of +ve arginine + lysine

core = two H2A-H2B dimers and a H3(_2)H4(_2) tetramer

wrapped DNA enclosed by superficial histone H1 at the entry/exit point of linker

amino acid tails protrude from each histone protein

55
Q

what does the compaction of chromatin mean for susceptibility to cleavage by DNase1?

what is the experiment elucidating this?

A

less susceptible

chicken embryos and Hb synthesis sites

in 20 hour old embryos, globin genes in precursor erythrocytes are insensitive to DNase1

as matures + Hb production begins, adjacent genes become susceptible to digestion

in tissues that don’t produce Hb, genes remain resistance to DNase1

56
Q

what are areas of high susceptibility to cleavage known as? why are they susceptible?

A

hypersensitive sites

have a scarcity of nucleosomes

57
Q

what is loosely packed chromatin called? what is its property?

A

euchromatin

have high transcriptional activity

58
Q

what group does histone acetyltransferase modify on the amino acid tails of histones? what is the effect?

A

adds acetyl groups from acetyl CoA to lysine residues on amino-terminal tails

lysine has NH4+ at physiological pH: acetylation = uncharged amide, reduces affinity of tail to DNA

histone complex loosens

59
Q

what does the now-acetylated lysine tail interact with? what is the effect?

A

bromodomains: acetyl lysine binding sites of 110 amino acids on regulatory transcription proteins e.g. TATA box bp and TAF1.

bind at H4

60
Q

what are chromatin remodelling complexes? what do they do?

A

contain bromodomains with helicase-resembling domains

use energy from hydrolysis of ATP to trigger conform changes in histone through shifting nucleosome position

renders genes more accessible and promotes binding of transcription complexes through protein-protein interactions

promotes gene expression

61
Q

multi-methylation of lysines on what histone is involved in compaction?

A

H3K9

62
Q

multi-methylation of lysines on what histone is involved in activation?

A

H3K4

63
Q

what can you covalently modify in the a to affect gene expression?
specific example?

A

lysine and arginine residues

trimethylation of lysine 27 on histone H3 = gene repression

64
Q

what covalent modification of DNA occurs? how?

A

methylation of cytosine and adenosine in the DNA

methyltransferase catalyse methyl group from (S-adenosyl methionine) SAM to C5 of cytosine in DNA

65
Q

what are the 3 types of methyltransferases? what are their roles?

A

DNMT1: maintains methylation from parent strand
DNMT3a, DNMT3b: drive de novo methylation of A and C

66
Q

how does presence of a methyl group impair gene expression?

A

impedes recruitment of transcriptional machinery thus prevents activation of genic regions (A. Dhar)

67
Q

what is the function of regulation of gene expression?

A

transcription of some genes over others allows for variation in protein synthesis across cells

repressor proteins prevent excess cell growth and suppress tumours

68
Q

what are the methods of gene expression regulation?

A
  1. acetylation of lysine on histones
  2. methylation of amino acids and DNA bases
  3. nuclear hormone receptors e.g. oestrogen
  4. DNA binding by transcription factors: homeodomains (zippers, h-t-h, cys-hys-zinc finger)
  5. activation domains on TFs
69
Q

how are the nuclear hormone receptors structured?

A

contain ligand-binding domain receptive to hydrophobic molecules e.g. oestrogen, thyroid hormone, steroid hormone

contains central DNA binding site w zinc domain

molecule to receptor binding = complex formed: transport to nucleoplasm then binds to 5’ - AGGTCAN-NNTGACCT - 3’ sequence in DNA

recruits coactivators (p160 family): SRC-1, GRIP-1

p160 family act as enzymes that upregulate chromatin remodelling: affecting gene expression

70
Q

what are the DNA-binding unit homeodomains?

A

h-t-h: helix inserted into major groove of DNA

leucine zipper: 2 alpha helices stabilised by leucine, involved in DNA site recognition

cys-hys-zinc-finger: tandems of small domains w 2 cysteine + 2 histidine bound to zinc + alpha helix which binds bps in major groove

71
Q

what do gene activator proteins do?

A

displace promoters from nucleosomes

as direct effect of TF or effect of interaction between regulatory proteins + general TFs

72
Q

what are the properties of active chromatin?

A

increased core histone acetylation: alters non-histone factors + all interaction types

phosphorylation of HI-type linker histones: causes histone to not associate with chromatin: creates binding site for reg. factors on DNA

increases incorporation of specific histone variants: affects nucleosome structure + packing

73
Q

what does the post-translational histone code determine?

A

gene expression levels

74
Q

what are the properties of inactive chromatin?

A

desphosph core histones

dephosph HI histones

diminished histone variants

subnuclear localisation of genes w silent genes near periphery

genes + molecules (7 decks)

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