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CBG > CBG04 > Flashcards

CBG04 Flashcards

1
Q

What size are RNAPs

A

500KDa

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2
Q

What is the structure of RNAP

A
  • two identical alpha subunits involved in binding rnap to dna
  • a flap to stop dna falling off
  • two beta sub units which form a clamp which the active site slips between
  • an omega subunit to stabilise the entire subunit
  • rudder to destabilise the rna/dna hybrid
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3
Q

What is an apoenzyme?

A

protein component of enzyme separate from prosthetic group, rewuires presence of conezyme to become active holoenzyme

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4
Q

What does the sigma subunit do?

A

apoenzyme needs to sigma to bind to dna as without sigma it cannot recognise the promoter region.
plays a similar role to initiator proteins - regulates transcription

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5
Q

what is the error rate for transcription in pro and euk?

A

10^-4

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6
Q

What is RNAPs proof reading mechanism?

A

modest,
incorrect ribonucleotide added rnap can back up and active site perform excision.
this resembles the reverse of polymerisation reaction except water inst4ead of pyrophosphate nis used and nucleoside monophosphates released

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7
Q

What are the 3 types of Eukaryotic RNAP

A

RNAP-1 = rRNA
RNAP-2= mRNA
RNAP-3 = tRNA
*structurally similar to each other and bacterial enxyme but transcribe different genes

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8
Q

What type of RNA do Archea and Eukaryotes have?

A

Archea - one RNAP similar to Eukaryotic

Prokaryotes - one kind

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9
Q

Describe RNAP-2

A

like bacterial RNAP but without sigma and with extra subunits and CTD tail
C terminal domain tail - very long repetitive tail that sticks out of enzyme

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10
Q

What inhibits RNAP-2

A

alpha amantin in death cap mushroom.

doesnt kill instantly but body cant make any more mrna so becomes weaker and dies

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11
Q

What is the promoter region in bacteria and eukaryotes?

A

Bacteria -Pribnow box tataat seq of 6 nucleotides

eukaryotes - tata box

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12
Q

What are the 3 sigma factors?

A

sigma 70- primary sigma factor - transcribes most genes in growing cells- keeps essential genes and pathways open
sigma 32 - heat shock sigma factor - turned on when sigma is exposed to heat - more sigma 32 is expressed, more likley to bind with rnap apoenzyme - expresses other heat shock proteins eg. chaperones, proteases, dna repair enzymes
sigma 38 = starvation, stationary phase sigma factor - helps adjust to stress

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13
Q

Describe transcription initiation in bacteria

A

1.sigma factor associates with core enzyme = rnap holoenzyme
2. complex adheres weakly to bacterial dna and slides along dna until it dissociates
3. when it reaches promoter region, sigma factor recognises and binds to it = pribnow box
4. rnap holoenzyme then opens up double helix to expose a short stretch of nucleotides on each strand.
5, doesnt used atp like helicase does, instead rnap and dna undergo a reversible structural change that results in a more favourable structure than binding.

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14
Q

What does transcription in eukaryotes require?

A

many different protein transcription factors. (unlike bacteria 1 sigma)
= general/basal transcription factors GTF
general because tehy are needed at nearly all promotors used by rnap11 denoted tf11 for rnap 11

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15
Q

What do GTF aid it?

A

positioning of rnap at promoter region.
pulling apart 2 strands of dna
releasing rnap from promoter into elongation mode once transcription has begun.

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16
Q

Describe transcription initiation in eukaryotes?

A
  1. TF11D binds to short DNA seq of T/A/s = TATA box -promoter region.
  2. section of TF11D that recognises promoter region is the TBP = tata binding protein
  3. binding of TF11D to TATA causes distortion in DNA of TAT box thought to serve as landmark - brings other factoes and rnap together to form transciption initiation complex. TIC
  4. After TIC formation on promoter DNA, RNAP11 must access template strand at transcription start point.
  5. TF11H which contains DNA helicase as one of its 9 subunits - hydrolyses ATP and unwinds DNA exposing template strand.
  6. TF11H phosphorylates CTD tail, allowing RNAP to go through a series of conformation changes that tighten its interaction with DNA
17
Q

What are the 3 main GTF’s involved in eukaryotic transcirption initiation.

A

TF11D - TBP, DNA distortion landmark for other GTFS
TF11H - DNA helicase, phosphorylates CTD for Conformational changes of RNAP to tighten interation
TF11B - recognises BRE (upstream of TATA) and positions RNAP11

18
Q

What does TF11B show homology with?

A

sigma factor - probably share a common ancestor.

19
Q

Describe Elongation in bacteria and eukaryotes?

A
  1. 1 strand of DNA acts as template
  2. Abortative initiation occurs - RNAP synthesizes the first 8-10 nucleotides by rocking on the promoter region- relatively inefficent process where RNA synthesizes and discards short RNA pieces.
  3. Promoter clearance occurs - a conformational change where RNAP weakens interaction with sigma and promoter region. And flap/clamp ttaps mRNA
  4. Elongation then begins RNAP, moves down - pyrophosphate is cleaved in 2pi with a massive change in delta G powering the reaction
20
Q

What is abortative initiation?

A

RNAP synthesizes the first 8-10 nucleotides by rocking on the promoter region- relatively inefficent process where RNA synthesizes and discards short RNA pieces.

21
Q

What is Promoter clearance?

A

a conformational change where RNAP weakens interaction with sigma and promoter region. And flap/clamp ttaps mRNA

22
Q

What does head of RNAP in eukaryotes contain?

A

histone acetyl transferase

which adds CH3COO- to lysine toe loosen the histone binding.

23
Q

What are the 2 types of termination in bacteria?

A

Rho dependent termination (P factor)

Rho independent termination (intrinsic termination)

24
Q

What is Rho?

A

a hexameric helicase protein that hydrolyses ATP-ADP and phosphate rotating as it does so. it is homologous to atp synthase.

25
Q

Where does Rho bind?

A

binds to RUT Rho Uilisation Site

a cytosine rich adn guanine poor region of 70-100 nucletoides

26
Q

What does Rho do?

A

binds to RNA and uses its ATPase activity to translocate along towards the 3’ end and unwind DNA/RNA duplex hybrid.

27
Q

What happens in Rho dependent termination?

A

Rho binds to RUT binding site

and the uses its ATPase activity to translocate along towards the 3’ end and unwind DNA/RNA duplex hybrid.

28
Q

What happens in Rho Independent/intrinsic termination?

A
  1. AT termination sequence = hair pin strucutre.
  2. pulls RNA transcript from RNAP active site.
  3. Uracil residue chain. = weak A-U binding.
  4. protein bound to rnap binds to stem and loop causes RNAP to stall
  5. coincides with poly U transcirption
  6. lowers energy of destabilisation
  7. RNAP and RNA dissociate.
  8. weakly bound poly U self terminates.
29
Q

What is polyadenylation?

A

Polyadenylation
addition of poly A tail to mRNA
unique to transcipts made my RNAP 11

30
Q

What are the 2 types of eukaryotic transcription termination and why is it difficult to describe?

A

Factor Dependent termination
Factor Independent termination
- it is the least understood of the 3 transcription stages

31
Q

Describe factor dependent termination

A

In RNAP1 = similar to rho dependent uses factors.
IN RNAP11 = CTD and CPSF - cleavage and polyadenylation specificty factor recognise poly A sequence and recruit other proteins to carry out cleavage and poly Adenylation
rnap continues to move along template generating second rna molecule associated with elongation complex.
- two models proposed
Allosteric model
Torpedo model.

32
Q

What is the allosteric model of factor dependent termination in RNAP11 eukaryotic transcription?

A

when transcription proceeds through the termination sewuences it causes disassemly of elongation factors and or assmebly of termination factors that causes a conformation change of elongation complex.

33
Q

what is the torpedo model of factor dependent termination in RNAP11 eukaryotic transciption?

A

torpedo model suggests that a 5’ to 3’ exonuclease degrades the second RNA as it emerges from the elongation complex.
RNAP released as exonuclease takes over.

CBG (19 decks)

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