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CBG PANT Replication, transcription and translation > CBG Lecture 4: Transcription > Flashcards

CBG Lecture 4: Transcription Flashcards

1
Q

how much more RNA than DNA do cells contain

A

20 times more RNA than DNA in cells

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2
Q

how much of cell is made up of RNA

A

5%

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3
Q

what is most common type of RNA

A

ribosomal RNA

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4
Q

what is transcription?

A

DNA -> RNA

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5
Q

what is error rate of transcription?

A

10-4 per nucleotide

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6
Q

what cofactors do both DNAPs and RNAPs contain

A

mg2+ cofactor, positive, so react with -ve phosphate charge on DNA

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7
Q

how much of RNA is mRNA?

A

mRNA is 5% of RNA

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8
Q

how large are RNAPs (kDa?)

A

500kDa

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9
Q

what is the machine used for transcription

A

RNA polymerase

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10
Q

which has a higher error rate - RNAP or DNAP?

why

A

RNAP has higher error rate due to it lacking a proof reading exonuclease

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11
Q

outline vague structure of RNAP + in bacteria

A

large multi subunit enzyme with a pincer like structure
have 5 core subunits
IN BACTERIA
composed of apoenzyme ( 5 core subunits) and sigma subunit, which makes up holoenzyme

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12
Q

how does bacterial RNAP differ from eukaryotic RNAP

A

bacterial RNAP has a sigma subunit

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13
Q

what is sigma subunit used for/by

A

used by bacterial RNAP to bind and melt promoter DNA - recognises the promoter

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14
Q

what do eukaryotic RNAPs require for initiation

A

basal transcription factores including the TATA binding protein

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15
Q

in depth structure of bacterial RNAPs

A

alpha 1&2 -> bind to DNA and stabilise
Beta&Beta’ -> form clamp, AS between them
w (omega) -> stabilise whole complex
beta flap -> traps mRNA
rudder -> destabilises DNA/RNA hybrid and allows sealing of transcription bubble

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16
Q

in what species is bacterial RNAP best characterised

A

Thermus aquaticus

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17
Q

what is an apoenzyme

A

part of bacterial RNAP structure, is the holoenzyme without the sigma unit

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18
Q

how many RNAPs do bacteria have? compare this to eukaryotic RNAPs

A

1

eukaryotes have 3 with the same 5 subunits

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19
Q

what are the different eukaryotic RNAPs, what do they each specialise in

A

RNAP1-ribosomal RNA
RNAP2 - mitochondrial RNA
RNAP 3 - transfer RNA
(1,2,3 = r,m,t)

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20
Q

in which organism is the speed of RNA transcription faster? E.coli or humans

A

E.Coli, by 30nt.s-1 (it is 50nt-1s-1)

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21
Q

what subunits are present in both bacterial and eukaryotic RNAPs

A

alpha1 &2
beta&beta’
omega

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22
Q

what is function of alpha 1&2 subunits in RNAP

A

bind and stabilise DNA

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23
Q

what is function of beta and beta’ subunits in RNAP

A

form a clamp with an active site between them

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24
Q

what is function of omega subunit in RNAP

A

stabilises the whole RNAP molecule

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25
Q

what is function of beta flap

A

traps mRNA

26
Q

how many RNAPs do Archaea have? what is this similar to?

A

1, similar to eukaryotic RNAP-2

27
Q

which chemical is RNAP2 v sensitive to?

A

alpha amanitin from Amanita phalloides

28
Q

outline structure of eukaryotic RNAP 2

A

like bacterial RNAP but no sigma SU, instead has extra subunits and a CTD (C-terminal domain tail)

29
Q

where does initiation of transcription begin

A

at conserved AT rich promoter sequences

30
Q

how is transcription initiated in bacteria

A

bac RNAP binds DIRECTLY to DNA via sigma factor (different factors have different specificities)

31
Q

what different bacterial sigma factors exist

A

sigma 70: general purpose

sigma s : stationary phase

32
Q

what is a Pribnow box
where is it found most
whats it recognised by

A

a consensus sequence for sigma 70 promoters which acts like a switch for sigma factor to recruit RNAP and therefore transcribe the Pribnow box
found in most E.coli genes

33
Q

Which sigma factor has a discrete setting, and which one is continuous

A

sigma 70 has differnt discrete settings and discrete modes of growth, recognising different promoters
when bac. reaches stationary phase, makes sigma S, clicks from one setting to the next`

34
Q

how is transcription initiated in eukaryotes

what do ALL eukaryotes require to initiate transcription via RNAP

A

requires several basal transcription factors and more complex promoters
All eukaryotes require a TATA BINDING PROTEIN (TBP)

35
Q

what is the sigma factor analogous to

A

TBP -> TATA binding protein in eukaryotes

36
Q

what do basal transcription factors do

A

act as a landing pad for RNAPs

37
Q

what is the most important component of TFIID (TF2D)

why

A

TATA binding protein, as it allows TF2D to recognise TATA box

38
Q

what does TATA binding protein do

A

allows TF2D to recognise TATA box

39
Q

what does sigma factor show HOMOLOGY to

A

TF2B

40
Q

what does TF2B do?

A

recognises BRE and positions RNAP2

41
Q

what is BRE

A

a transcription factor 2 response element (TF2B response element)

42
Q

what does TF2H doe

A

melts DNA and phosphorylates CTD

43
Q

what does TF2D do

A

recognises TATA box via TATA binding protein

44
Q

what are the main basal transcription factors that initiate transcription in eukaryotes

A

TF2D -> recognises TATA boc via TATA binding protein
TF2H -> melts DNA and phosphorylates CTD
TF2B -> recognises BRE (its response element) and positions RNAP2

45
Q

what happens to RNAP to allow it to start transcription

A

it starts transcription when phosphorylated

46
Q

why are eukaryotic genes more continuous when comparing to bacteria

A

they have multiple promoter sequences eg.BRE DPE CAT and by mixing and matching you get various degrees of tuning, therefore more boxes = more recruitment of RNAP = more RNA = more protein

47
Q

what are the following:
BRE
CAT
DPE

A

multiple promoter sequences involved in initiation of eukaryotic transcription

BRE: TF2B response element
INR: initiator sequence
DPE: Downstream promoter element

48
Q

outline sequence of events in initiation of eukaryote transcription

A
  1. TBP binds -30 upstream of sequence, binds and melts DNA
  2. TF2D recognises TATA box via TBP
  3. TF2B recognises BRE and positions RNAP2
  4. TF2H melts DNA and phosphorylates CTD (C-terminal Domain) using a kinase
  5. when phosphorylated, RNAP leaves behind lump of basal transcription factors and begins transcribing
49
Q

why is transcriptional elongation in eukaaryotes harder than in bacteria

how is this overcome?

A

because nucleosomes must be remodelled as histones are present
this is overcome by:
histone acetyltransferase acetylates lysine and loosens histone binding
allows RNAP to navigate its way around

50
Q

why can RNA polymerases start an RNA chain without a primer unlike DNAP?
compare error rates in both DNAP and RNAP

A

because transcription need not be as accurate as DNA replication
Unlike DNA, RNA does not permanently store genetic information in cells.
RNA polymerases make about one mistake for every 104 nucleotides copied into RNA (compared with an error rate for direct copying by DNA polymerase of about one in 107 nucleotides), and the consequences of an error in RNA transcription are much less significant than that in DNA replication.

51
Q

compare error rates in both DNAP and RNAP

A

RNA polymerases make about one mistake for every 104 nucleotides copied into RNA (compared with an error rate for direct copying by DNA polymerase of about one in 107 nucleotides),

52
Q

what is abortative initiation

A

the initial RNA synthesis where RNAP rocks on promoter and ‘poops’ 8 nucleotide fragments
then promoter clearance (conformational changes allowing elongation)

the RNA polymerase holoenzyme (apoenzyme&sigma factore) forms and then locates a promoter.
the polymerase unwinds the DNA at the position at which transcription is to begin, and begins transcribing

53
Q

what happens in transcription elongation

A

during elongation NTPs are incorporated into the mRNA
abortative initiation occurs
then promoter clearance

54
Q

what is promoter clearance

A

gets over “hump” of making c.10nt of RNA
conformational changes
sigma factor dissociates (in bac) / TFs left behind on promoter in euks

55
Q

how is termination achieved in proks

A

rho factor and secondary mRNA structures

56
Q

how is termination achieved in euks

A

polyadenylation

and secondary mRNA structures

57
Q

how does transcriptional elongation occur in bacteria
give word eqtn for this
what is driving the rraction

A 
NTPs are incorporated into the mRNA
jaws down, flap up,elongate piece of DNA
in bacteria, sigma factor is no longer associated with RNAP and falls off
1.Abortative initiation
2.promoter clearance
RNAn+NTP -> RNAn+1 +2Pi
 a negative DG which is driving reation
58
Q

what is polyadenylation

where does it occur

A

PROKS: addition of polyA tail to mRNA which contains lots of AMP
eg. stretch of RNA that only ha adenine bases
it produces mature mRNA for translation

59
Q

what is a rho factor

where does it occur

A

in PROKS:
rho factor is a hexomeric helicase
hydrolyses ATP to ADP and Pi as it does it rotates and drills way down DNA and u RNA
rho binds to the transcription terminator pause site; an exposed region of ssRNA
each subunit has an RNA binding domain and atp hydrolysis domain

60
Q

what are the 2 types of transcriptional termination in proks?
what determines which route transcription termination takes

A
  1. rho dependent
  2. rho independent (intrinsic)

the RNA primary and secondary structures determine how its treated in cell and what happens to it

61
Q

how can mRNA change its secondary structure in order to terminate transcription

A

Self-complementary sequence forms hairpin, disrupts RNAP’s flap
Poly-U weakly bound to DNA

62
Q

outline bacterial RNAPs cycle

A
  1. sigma factor binds apoenzyme
  2. holoenzyme begins initiation - binds DNA via sigma
  3. melts DNA - sigma dissociates
  4. abortative initiation
  5. promoter cleaANCE
  6. elongation
  7. hairpin or Rho begins termination

CBG PANT Replication, transcription and translation (10 decks)

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