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Gene to function - Eukaryotes > Lecture 16 - Basal transcription > Flashcards

Lecture 16 - Basal transcription Flashcards

1
Q

What is the central dogma of gene expression?

A

DNA is transcribed into RNA and translated into protein

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2
Q

Where does most regulation occur in gene expression?

A

at the transcription level

-exceoption is viruses

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3
Q

What ar a few examples of where transcription control is used?

A
  • during cellular differentiation
  • in response to stress
  • in response to hormonal stimulation
  • changes in nutirent status or in response to growht factor signals
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4
Q

Give examples of what can happen when transcription regiulation goes wrong?

A

Cancer

  • retinoblastoma caused by a Rb loss fo funtion mutation
  • recessive gene
  • loss of sight amogst other things

antp mutant drosophila
-legs growing out of where antenna should be

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5
Q

What are the three stages of transcription?

A

Initation
Elongation
Termination

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6
Q

What is the structure of the E.coli RNA pol (prokaryotic)?

A
  • very simple
  • only one form of RNA pol
  • 4 subunits: β, β’, 2Xα
  • σ factor binding confers DNA specifiicty (holoenzyme)
  • core RNA poly has no ability to specifically bind DNA
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7
Q

How were the 3 eukaryotic DNA pol identified

A
  • took sea urchin nuclear extract and manipulated so that the RNA contains radioactive UMP(instead of uracil)
  • did purification step [used, DEAD sephadex column followed by an ion exchange column for negative charge (to isolate the factor that binds DNA)
  • took fractions as increased the salt (ammonium sulfate)
  • added phosphor and collected fractions and tested for the inclusion of radioactive UMP

Would have expected one peak of activity like the E.coli polymerases
-however actually got 3; RNA pol I, RNA pol II and RNA pol III

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8
Q

What are the genes transcribed by the three RNA polymerases?

A

RNA pol I: Large mRNA
RNA pol II: mRNA, U1-U5 snRNA
RNA pol III: small rRNA, tRNA, U6 snRNA, VA1 RNA and other small RNAs of unknown function

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9
Q

Which RNA pol is involved in transcription of RNA polymerase III?

A

RNA pol II

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10
Q

What are the yeast RNA pol subunits?

A

Core subunits
Common subunits
Specific subunits (uniquie to RNApol but not part if the core subunits)

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11
Q

What are the features of RNA pol II?

A

-large, multi-subunit complex of proteins
-14 subunits in yeast
[3 largest are similar to bacterial RNA pol subunits, 2 largest carry the catalytic site]
-largest subunit has a carboxyl-terminal domain (CTD) that consists of a repeated sequence of 7 amino acids, phosphorylation of this domain is important for transcription initiation and downstream processing

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12
Q

What is the sequence of amino acids in RNA pol II CTD?

A

YSPTSPS

contains tyrosine, threonine and serine - sites for phosphorylation

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13
Q

Compare the euakryotic to the prokaryotic mode of transcription

A

Pro
-E.coli genome is circular, super coiled DNA molecule lacking any higher order chromatin structure
-Therefore the first step in gene expression (promoter recognition and DNA binding) faces less obstables
-repression is more necessary
= Non-restrictive transcription
Eu
-DNA is packaged into higher order chromatin structure
-keeps genes inactive by preventing RNA pol from accessing the promoter
-acitivation more necessary
=restrictive transcription

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14
Q

What are the regions of the RNA pol II promoter?

A

A core promoter region
-located proximal to, potentially overlapping with, the start site of transcription and is suffient for low level of transcription in vitro
A regulatory region
-usually distal to the start site for activator or repressor binding

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15
Q

What does the promoter determine?

A

When RNA pol will bind

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16
Q

What are the properties of the RNA pol II core promoter?

A

Start point (start of transcription not translation)
-first base of mRNA (+1 position) to be A and flanked on either side by pyrimidines (C and D)
-hard to determine position
TATA box
-~25-30 bases upstream of the start point
-found in most genes in eukaryotes
-equivilent of the -10 seq in bacterial promoters (Pribnow box)
-AT rich consensus sequence often surrounded by GC rich sequence
-TATA box is the initial DNA target site for the assembly of the basal transcription complex in most promoters

17
Q

What is an initiator element?

A

instead of a TATA box, some RNA pol II promoters contain an alternative promoter element
e.g. lymphocytes in TET gene

18
Q

What RNA pol II require to initiate rtanscription?

A

-auxillary factors (transcription factors)

19
Q

What is TFIID?

A
  • binds to theTATA box
  • compoased of TBP and TAFs
  • TBP(TATA box binding protein) recognises the TATA box
  • TAFs (TBP-associated factors)
  • TBP bends DNA ~80* and forces open the minor groove
  • melts DNA
  • also provides a saddle structure for the loading of other GTFs
20
Q

How was TFIID identified?

A

-ran a negatively charged column
-took different concentrations of salt to elucidate DNA/DNApol binding proteins; A - 100nm, B - 350nm, C - 500nm, D - 1mole
-isolated the binding proteins and named
e.g.
TFIID isolated in the D fraction

21
Q

What is the process of assembly of the basal transcription process?

A
  1. TFIID binds to the TATA box, facilitated by TFIIA
  2. TFIID-DNA complex is recognised by TFIIB
  3. TFIIB binding recruits RNA polymerase II along with its assocaited factor TFIIF
  4. when RNA pol II joins, the CTD binds to TBP
  5. subsequently, TFIIE and TFIIH (multisubunit complexes join the complex, leading to promoter clearance and melting
  6. TFIIH has helicase and kinase activity, phosphorylation of CTD caused RNA pol II to no longer be tethered to the TBP and RNA pol II can now move off
  7. the phosphorylation of CTD also induces a conformational change that releases TFIIE from polII and TFIIH
  8. once TFIIE is displaced, the TFIIH helicase acts to unwind the DNA template
  9. PolII and TFIIF move off down the gene leaving TFIIA and TFIID bound at the promotor
22
Q

What is the process of promoter clearance and melting?

A

TFIIE and TFIIH involved

  • TFIIH has helicase and kinase activity, phosphorylation of CTD caused RNA pol II to no longer be tethered to the TBP and RNA pol II can now move off
  • the phosphorylation of CTD also induces a conformational change that releases TFIIE from polII and TFIIH
  • once TFIIE is displaced, the TFIIH helicase acts to unwind the DNA template
23
Q

Can the basal transcriptional complex produce any expression of genes without the help of stimulatory transcription factros?

A

Yes. The basal transcriptional complex can produce only a low rate of transcription

24
Q

What is required for significant levels of expression?

A

Basal transcriptional complex, and stimulatory transcription factors that bind to DNA sites distinct from where basal transcriptional complexes assemble

25
Q

What are the features of the stimulatory transcription factors that bind DNA to produce significant levels of expression>?

A
  • bind to DNA distinct from when the basal factors assemble
  • DNA sites can be short elements located within or adjaceent to the gene promoter, or can act at a distance
  • efficiency and specificity depends on the particular combination of sequence elements to which regulatory sequences bind
  • sequences may be common and found in a wide variety of promoters or they may be specific, to allow for tissue specific expression or inducability under certain condiditons

Gene to function - Eukaryotes (13 decks)

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