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CMB2001 - Control of Eukaryotic Gene Expression > Lecture 1 - Transcription Initiation in Eukaryotes > Flashcards

Lecture 1 - Transcription Initiation in Eukaryotes Flashcards

1
Q

what is gene expression?

A

process by which information in genes (DNA) is decoded into protein

multistep process

highly regulated

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2
Q

what is transcription?

A

transfer of genetic information from dsDNA to ssRNA (mRNA)

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3
Q

how does transcription in prokaryotes work?

A
  1. holoenzyme recognises and binds to sequences upstream of the promoter - this is a closed complex
  2. during transcriptional initiation the DNA becomes melted over the transcription start site and becomes locally single stranded - transcription bubble
  3. RNA polymerase now has access to a ss template & makes mRNA copy
  4. after 10 copies sigma70 is released and process starts again
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4
Q

what is the holoenzyme in prokaryotes?

A

core RNA polymerase + sigma70

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5
Q

what are prokaryotic promoters?

A

cis acting DNA regulatory elements through which transcription is initiated & controlled

well conserved sequences located -10 and -35 from the start site

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6
Q

how does the location of the promoter sequence affect the RNA polymerase binding in prokaryotes?

A

the closer the promoter sequence is to the consensus, the more effectively RNA polymerase binds and initiates transcription

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7
Q

what is the core region in eukaryotic promoters?

A

basal region

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8
Q

what are the different core region elements in eukaryotic promoters?

A

TATA box is located 30 bases upstream of the start site

initiator element is located above the start site

downstream promoter element (DPE) is 30 bases downstream of start site

core regions don’t tend to have all of them - have 1 or 2

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9
Q

what are core promoter elements associated with?

A

regions with a high frequency of CG sequences: CpG islands

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10
Q

why is the state of methylation in CpG islands important for promoter function?

A

methylation of CpG islands is associated with silencing - transcription is ‘switched off’

in mammals most C residues followed by G are methylated to 5-methyl C

generally C resides in CpG islands escape methylation - hypomethylation

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11
Q

what are the 2 sections of the regulatory region in eukaryotes?

A

distal or proximal

elements are either close or far away from the core region

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12
Q

what are the activator binding sites in the regulatory region?

A

UAS and the enhancer

  • UAS is proximal
  • enhancer is distal
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13
Q

what are the repressor binding sites in the regulatory region?

A

URS and the silencer

  • URS is proximal
  • silencer is distal
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14
Q

what does UAS and URS mean?

A

upstream activating sequence

upstream repressing sequence

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15
Q

what tools are used to identify promoter elements?

A

sequence comparison

reporter analysis

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16
Q

what is sequence comparison?

A

identification of the TATA box

line up sequences and look for sequences that appear with high frequency

can tell you about some sequence elements but doesn’t give any idea of function

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17
Q

what is reporter analysis?

A

uses reporter genes - encode proteins whose levels can easily be measured

are often enzymes

18
Q

what enzymes can be used in reporter analysis?

A

GFP - green fluorescence protein

luciferase - in the presence of ATP and the right substrate give off light

lacZ - beta-galactosidase from E.coli

19
Q

how does reporter analysis work?

A
  1. reporter gene has its native regulatory sequences taken away and is put under the control of the promoter of the regulatory sequence interested in - this drives the reporter gene
  2. clone that into a plasmid and transfect it into cells
  3. if the regulatory sequence is active it will drive transcription of the reporter gene which will be converted into protein which can then be measured
  4. gives you a ‘report’ on whether the promoter is active
20
Q

what is promoter bashing?

A

identification of promoter elements using 5’ deletion series of reporter constructs

allows you to functional define the important regions in the promoter

21
Q

how does promoter bashing work?

A
  1. make a variety of versions of the promoter - truncate pieces away from the 5’ end
  2. make a series of plasmids with different promoters
  3. transfect these into cells
  4. measure the amount of activity
22
Q

what can reporter analysis be used to identify?

A
  • when a gene is expressed
  • where it is expressed
  • what signals it responds to
  • what factors and sequences control its expression
23
Q

what are the 3 major eukaryotic RNA polymerases?

A

RNA pol I
RNA pol II
RNA pol III

24
Q

what do the different RNA polymerases do?

A

RNA pol I controls rRNAs

RNA pol II makes mRNA. snRNAs and miRNAs

RNA pol III responsible for tRNAs

25
Q

what does RNA pol I do?

A

controls rRNA

in the nucleolus

26
Q

what does RNA pol II do?

A

makes mRNA. snRNAs and miRNAs

in the nucleus

27
Q

what does RNA pol III do?

A

responsible for tRNAs

in the nucleus

28
Q

what are the GTF?

A

general transcription factors

role is to recruit RNA polymerase to the promoter to start transcription

29
Q

what are the different general transcription factors?

A

TFII-A/B/D/E/F/H

30
Q

how is the pre-initiation complex (PIC) assembled?

A
  1. assembly of TFIID over the TATA box is stabilised by TFIIA
  2. TFIIB can then bind and recruit RNA pol II
  3. TFIIF comes with the RNA pol II to allow assembly of TFIIE and TFIIH
  4. TFIIH allows initiation of transcription
31
Q

general order of the PIC

A 
D
A
B
RNA pol II 
F
E
H
Transcription
32
Q

what does TFIID do?

A

binds to the TATA box

recruit TFIIB

33
Q

what does TFIIA do?

A

stabilises TFIID binding

anti-repression function

34
Q

what does TFIIB do?

A

recruiters RNA pol II-TFIIF

important for start site selection

35
Q

what does TFIIF do?

A

stimulates elongation

destabilises non-specific RNA pol II-DNA interactions

36
Q

what does TFIIE do?

A

recruited TFIIH and modulates its activity

37
Q

what does TFIIH do?

A

promotes melting and clearance

CTD kinase activity

DNA repair coupling

38
Q

what happens once TDIIH has inited transcription?

A

helices activity of TFIIH separates the template strand at the start-site to form open complex

as pol II starts transcribing (promoter clearance) it is phosphorylated on the C-terminal domain (CTD)

39
Q

what happens to the general transcription factors once transcription has started?

A

TFIIF moves down template with pol II

TFII-B/E/H are released

TFII-D/A stay behind for next round of transcription

40
Q

structure of TFIIH

A
  • composed of 9-10 subunits
  • divided into 2 parts - CORE + CAK
  • is the central RNA pol II TF
  • made of TATA binding protein (TBP) and TBP associated factors (TAFs)
  • contains an ATPase helices called XPB that plays a role in promoter melting
41
Q

structure of TFIID

A
  • trilobular structure

* TBP is a molecular saddle in the middle

42
Q

what do TAFs do?

A

TBP associated factors (TAF)

  • promote interaction of TFIID with the basal promoter
  • interact with activators

CMB2001 - Control of Eukaryotic Gene Expression (10 decks)

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