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BIOL2202 > 3 - gene regulation > Flashcards

3 - gene regulation Flashcards

1
Q

how is transcription initiated?

A

• different sigma factors compete to bind to the core polymerase
—–> binds to -35 region = tight complex
—–> binds to -10 region (AT rich) = open complex
• binding of sigma factors restricts polymerase from binding to specific promoters
• RNA pol. slides along DNA and binds tightly if it matches the sigma factor
• sigma factor released and transcription initiated

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2
Q

what are the sigma factors in E.coli?

A

1) σ(D) = “housekeeping genes”
2) σ(S) = starvation / stationary phase
3) σ(H) = heat shock (also triggered by cold, osmotic pressure, etc)
4) σ(E) = extreme heat shock
5) σ(I) = iron deficiency
6) σ(F) = flagellar genes (developmental)
7) σ(N) = nitrogen deficiency

1 = high affinity for RNA pol, high abundance (constantly expressed)
2-7 = low affinity for RNA pol, low abundance
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3
Q

what is the stringent response?

A

under good nutritional conditions:
• σ(D) is bound to RNA polymerase
• 60-80% of genes are transcribed
• transcripts very stable

under poor nutritional conditions:
• σ(S) replaces σ(D)
• normal "house-keeping" supressed
• 10% genes are overexpressed 
• expressed genes necessary for during starvation = cell survival
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4
Q

what is the functional role of alarmone / ppGpp / magic spoT?

how is it synthesised and degraded?

A
  • ppGpp binds RNA pol to reduce it’s affinity for σ(D)
  • allows σ(S) and other stress σ-factors to compete for binding
  • ppGpp is synthesised by relA and spoT
  • —> RelA + uncharged tRNA (i.e. in aa starvation)
  • —> spoT - glucose (i.e. in glucose starvation)

• ppGpp degraded by spoT
—-> spoT + glucose (i.e. in glucose abundance)

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5
Q

how does lac operon function based on the available metabolites?

A

during glucose or aa starvation:
• ppGpp produces by relA or spoT
• σ(D) released from core RNA polymerase
• σ(S) binds = starvation response holoenzyme
• RNA polymerase will bind to -35 & -10 regions of lac promoter —> primed for de-repression by allolactose

if glucose is unavailable:
• cAMP is a specific glucose starvation signal
• cAMP binds CRP (cAMP receptor protein)
• CRP + σ(S) promotes strong binding of holoenzyme
—-> facilitates transcription

if lactose is available:
• allolactase signals lactase availability
• allolactase binds to lac operon repressor to prevent inhibition of lac operon transcription

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6
Q

how is transcription regulated by peptide hormones?

A
  1. insulin binds receptor in membrane of target cell
  2. activates signalling pathway on cytoplasmic face of receptor
  3. info transduced through pathway until it reaches nucleus
  4. TF modified to initiate transcription of DNA
  5. transcript processed and transported to cytoplasm
  6. mRNA translated into proteins
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7
Q

how is transcription regulated by steroid hormones?

A
  1. steroid hormone enters cell and combines with a receptor protein
  2. hormone/receptor complex binds to a response element in the DNA
  3. bound complex stimulated transcription
  4. transcript processed and transported to cytoplasm
  5. mRNA translated into proteins
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8
Q

how does heat shock induce transcription?

A

• cell under heat stress phosphorylates HSTF
• housekeeping genes suppressed within 300 seconds
• response genes induced within 30 seconds
—–> RNA initiates transcription of heat shock genes
transcription halted because RNA polymerase gets tethered to DNA
—–> transcription re-initates when required
• induced proteins, e.g. hsp70, facilitate restoration and reuse of proteins, stabilisation of membranes and changes to gene expression

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9
Q

name 6 factors which induce longevity

A

1) dietary restriction
• increase lifespan 20-30%
• ­↑ longevity
• ↓ growth & reproduction

2) ↓ insulin signalling
• results from cellular glucose deficit
• ­↑ longevity
• ↓ growth & reproduction

3) AMP kinase signalling
• results from energy depletion (i.e. ↑AMP/ATP ratio)
• ­↑ longevity
• ↓ growth & reproduction

4) amino acid signalling
• results from aa deficit
• ­↑ longevity
• ↓ growth & reproduction

5) ↓ mitochondria function
• results from energy depletion
• ­↑ longevity
• ↓ growth & reproduction

6) ↓ temperature
• ­↑ longevity
• ↓ growth & reproduction
• ↓ metabolic rate

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10
Q

why is C.elegans used for longevity research?

A

1) short lifespan ~3 weeks
2) small –> easy to culture
3) self-fertilising hermaphrodites –> easy to mutate and inbreed
4) fertile! ~300 offspring

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11
Q

describe the cellular signalling in C.elegans

A

peptide signalling:
• insulin/growth factor receptor at cell surface
• phosphorylation cascade
• terminal target is a gene regulatory protein
• phosphorylation prevents nuclear entry
• prevents activation of starvation genes (in well fed state)

steroid hormone:
• nuclear hormone receptor in cytoplasm
• hormone binding allows:
– entry to nucleus 
– gene regulation
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12
Q

describe the structure of the regulatory region

A

CORE PROMOTER
• RNA pol. assembly site
• 100-200 bp of DNA
• immediately upstream of transcription start

PROXIMAL ELEMENTS
• site of binding for activator proteins
• few 100 bp of DNA
• immediately upstream of promoter

DISTAL ELEMENT (ENHANCER)
• site of binding for special TFs 
• spread across 1000-10,000 bp of DNA
• sometimes in introns and rarely downstream of transcribed gene (function in either orientation)
• actual binding sites vary from 4-8bp
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13
Q

describe the structure of transcriptional proteins

A 
• basal TFs
– A B D E F
– 25 subunits in total (i.e. 25 different proteins assembled
• RNA polymerase II
– >30 subunits
• pre-initiation complex
– basal TF + RNA polymerase II
– transcription only begins when polymerase is phosphorylated
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14
Q

how are basal transcription factors assembled?

A
  • TFIID binds to TATA box & bends DNA sharply • Other basal factors assemble
  • AT rich = weak bonds, therefore sharp bending separates DNA strands apart
  • RNA polymerase II holoenzyme binds to TFIID
  • bent DNA promotes ‘melting in’ of RNA polymerase II
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15
Q

what is the mediator complex?

A

mediator complex binds to activator proteins at proximal elements (the DNA sequence)

~ 30 subunits

special TFs which bind to distal elements interact with mediator to promote assembly of RNA pol. II

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16
Q

what is the structure of the transcription factor

A

TF has a modular structure

1) DNA binding domain
– recognises 4-8bp sequences

2) dimerisation domain
– most TFs are inactive as monomers
– homo- or hetero-dimer formation
– tremendous combination specificity

3) activation domain
– interacts with subunits of mediator or RNA Polymerase II

17
Q

how are genes most commonly varied?

A

genes are most commonly altered via their expression

transcription factors alter expression levels of entire suites of interacting genes, making them highly important in evolution

hormones can also alter suites of genes

18
Q

what is the structure of chromatin?

how is it activated/inactivated?

what else is induced by H3K9 methylation?

A

chromtin = DNA + protein

nucleosomes = 8 histone proteins

  • –> H2aH2b + H3H4 dimers form octamer
  • —> H1 joins = very compact

for chromatin activation, nucleosomes must be accessible

• H1 released via phosphorylation to release coil
• H3 modified by acetylation/methylation so nucleosome can switch between active and inactive
– K9 acetylation = active
– K9 methylation = inactive
– K4 methylation enhances active state

active = histones able to be removed

  • H3K9 methylation can induce DNA methylation
  • DNA methylation useful for long term changes in gene expression (e.g. developmental transitions in plants)

permanent gene inactivation = chromatid inactivation via histones + methylation (hides DNA away)

19
Q

how are viruses and transposons inactivated/activated

A
  • viruses and transposons are dispersed throughout the genome
  • they are inactivated by methylation
  • become reactivated under host stress
  • may lead to an advantageous mutation but lethal if they disrupt a vital gene
20
Q

is methylation inherited?

A

methylation from previous generation erased prior to embryo implantation
i.e. no methylation is inherited from parents

exception: parents under severe stress (starving) —> epigenetic info passed —> offspring more adapted to cope

re-methylation coincides with developmental progression

21
Q

how is methylation affected by diet?

A

mother’s diet during development can influence epigenetics of child

supplement diet with methyl donors (folic acid, B12, choline, betaine) to increase methylation

different methylation → different gene expression → different phenotype

22
Q

what is DNA imprinting?

A

imprinting = 30-200(~1%) of genes are differentially inactivated by methylation in gametes

imprinted genes = mutant phenotype that depends on parent of origin

methylation can also affect the penetrance of the phenotype

imprinting erased early in gamete cell formation

during formation of oocytes → female imprinting established
during formation of sperm → male imprinting established

23
Q

what are the four types of methylation?

A

1) DYNAMIC GENE REGULATION
– mostly erased in early embryogenesis
– re-established during lifetime

2) IMPRINTING
– erased and re-established early in gamete formation

3) SUPPRESSION OF TRANSPOSONS
– by piRNA signalling
– maintained indefinitely but erased under stress

4) X-CHROMOSOME INACTIVATION
– cell-by-cell decision in early female embryo
– X from male inactivated in placenta (in mice)

24
Q

what is co-suppression?

what is transgene suppression?

A

endogenous genes are suppressed together

RNA-mediated

this suppression can spread to transgenic tissue

these suppressed genes are methylated

transcripts are inhibited or degraded → depends on which piece of gene was inserted into transgenic organism

25
Q

what are micro RNAs?

A

micro RNA, miRNA = small non-coding RNA molecule

functions in RNA silencing and post-transcriptional regulation of gene expression

bind to 3’ end of transcript
– imperfect hybrids inhibit translation
– perfect hybrids ↑ transcript degradation

26
Q

what is RNA interference?

A

RNA interference, RNAi

RNA molecules direct proteins to degrade homologous mRNA

results in gene suppression

RNAi is also a mediator of co-suppression

27
Q

what is RISC?

A

RISC = RNA induced silencing complex

RNA directs RISC to target nucleic acid

target identified by complementary base-pairing

28
Q

mechanism of post-transcriptional regulation by RNA interference

A

1) large dsRNA is diced into small dsRNAi’s
2) the small RNAis and proteins assemble into ribonucleoprotein particles
3) the small interfering RNA in a ribonucleoprotein particle is unwound to produce an RNA-Induced Silencing Complex (RISC)
4) the RISC targets a sequence in a messenger RNA that is complementary to the RNAi
5) the RISC’s RNAi base-pairs with its target in the messenger RNA

6)
a) if perfectly base-paired, the mRNA is cleaved and the mRNA is degraded
b) if imperfectly base-paired, translation of the mRNA is arrested and polypeptide synthesis from the mRNA is repressed

29
Q

what are regulatory RNAs and what is their function?

A

regulatory RNAs = small non-coding RNAs which regulate cellular functions

RNA molecules identify RNA/DNA sequences by complementary base pairing and direct proteins to target sequences

these RNA signals can travel between cells

RNA also signals to germ-line to control inheritance of epigenetic state

BIOL2202 (6 decks)

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