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Genetics > Basic transcription mechanisms, (lecture 2 and 3, not finished) > Flashcards

Basic transcription mechanisms, (lecture 2 and 3, not finished) Flashcards

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Biology 101 flashcards
1
Q

coding strand?

A

5’-3’, also calles non-template, indentical to RNA transcript

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2
Q

template strand?

A

3’-5’

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3
Q

promoter vs TSS

A

promoter: - DNA region/sequence where the RNA Pol binds

- it lays -35 to -1/+1 from the TSS (transcription start site)

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4
Q

reaction catalyzed by RNA Pol?

A

hydrolysis of pyrophosphates of unbound NTPs

driven by negative energy of 3’ oxygen atoms

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5
Q

phases of transcription? what happens

A

initiation: RNA Pol binds, unwinding, short chains are polymerized
elongation: RNA transcipt is made
termination: RNA Pol and RNA trancript are released

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6
Q

sigma factor

A

only in procaryotic transcription!
ensures specific binding of the RNA Pol
enables initiation (not binding)

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7
Q

elements of an promoter

A

(never present all at once - modularity)

  • -35 and -10/TATA/Pribnow Boxes
  • elements affecting efficiency (UP, Ext; Dis)
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8
Q

distance between the two promoter bosxes and why

A

in 90 % of teh cases: 16-18 bp

important but why???

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9
Q

general function of consensus sequences

how could they be found

A

several regions of the protein sigma factor and CTDs of the alpha subunits of the RNA Pol bind there

crystal structure analysis (X ray crystallography) of the bacterial RNA Pol holoenzyme bound to promoter DNA

but: not a every day method although it reveals a detailed view
solution: footprinting

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10
Q

footprinting:

explain

resolution?

A
  • label DNA with radioactive nucleotides - (32)P
  • introduce nicks (Schnitte) by DNAse
  • Gel-electrophoresis
  • footprinkt on gel shows where the protein was bound
  • simoultanious sequencing rection

2 reactions:

1 without protein = control -> fragments of every length

2 with protein: different pattern

asymmetric digestion: binding site close to labelled site
why???

(Lecture 2 - 23)

single nucleotide resolution

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11
Q

how can you label DNA ends?

A

Klenow fill-in reaction
(Klenow enzyme)

make sticky ends (by PCR?)
(create 5’ overhang at the primer) and fill it up with radioactive nucleotides (a-phosphate must be labelled, not the gamma!)

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12
Q

what is the footprint?

A

it is the break in the ladder distribution of DNA fragments, caused because the binding protein protected the DNA at this specific site

you can run a sequencing reaction at the same time -> identificatiob of the binding site

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13
Q

key idea of sanger sequencing

A

use of dideoxynucleotides (ddNTPs):
- they cause termination of elongation
. they are labelled
-> ends are always labelled

4 separate reactions: each containing 3 normal nucleotides and aditionally 1% ddNTP + 99% dNTP

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14
Q

what can you detect by DNA footprinting>?

A

DNA protein interaction

binding site of the protein

shape changes of the binding protein (it can bind larger or smaller parts of DNA in course of the time)

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15
Q

how can you detect unwinding?

A

treat witha reagent which only gets access to a single strand
e.g. KMnO4

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16
Q

why is -10 rich of which nucleotides?

A

rich of T and A because they only bind 2 hydrogen bridges each bp

important because during initiation the double strand needs to be unwound/melted

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17
Q

summary prokaryotic promoter

A
  1. Modular
    2.Consensus sequence
    3.Most important: -35 and -10 box
    4.Mutations may affect:
    sfactor and polymerase binding
    DNA unwinding
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18
Q

specific function of the 2 promoter boxes?

what does mutation impair?

A
  • 35: formation of closed complex= binding of RNA POL

- 10: the former and/or formation of open complex = unwinding of ds

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19
Q

stages of initiation

type of initiation?

A
  • closed binary complex: promoter recognition
  • open complex: melting of DNA, “jaws close”
  • ternary complex: RNA Pol + DNA + first RNA nucleotides, abortve initiation

abortive initiation: short RNAs formed and released
RNA polymerase stays on promoter
„DNA scrunching“

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20
Q

transition to elongation problems? solution?

A

1) initiation requires tight binding to specific seq whereas elongation requires binding to all encountered sequences
2) sigma factor (which mediates the specific binding to the promoter) blocks the exit channel for RNA

sigma factor is released -> TEC (transcription elongation complex) or GEC?

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21
Q

what’s the catalytic cofactor in the RNA Pol and why?

A

Mg 2+
because of its positive charge?:

  • facilitates attack of 3‘ OH
  • stabilizes negative charges of transition state
22
Q

model involved in chemistry of adding NTPs?

A

Brownian ratchet model:

the transition state of elongation is stabilized (can not go back)

the reactions before and after the intermediate EC can happen more easily forwards but can hardly happen backwards (like a ratchet)

23
Q

new method to observe base.-stepping

A

optical tweezers(=Pinzette):

small beads can be trapped in highly focused laser beams,
position of the beads can be monitored with high precision

DNA and RNA Pol can be bound to the beads, so every movement can be monitored

resolution: addition of 1 NTP

24
Q

Blocks of transcription?

solution?

A
  • RNA hairpin structures
  • misincorporation

RNA Pol has a intrinsic cleavage activity (pyrophosphorolysis)
-> new 3’-OH end

25
Q

termination of transcription

classes?

A

1) intrinsic terminators (no other factors required)

2) rho (r)dependent terminators

26
Q

why is it experimentally difficult to find termination sites?

A

Often difficult to find the termination site:

1) in vivo, the primary transcript gets cleaved or partially degraded
2) in vitro, experimental conditions influence termination capacity
- > if both approaches find the same, probably the true site…

27
Q

intrinsic termination

A

through hairpin structures in the transcribed RNA (which means the termination sequences are in the transcribed region of the DNA)

hairpins are caused by 2 G-C rich regions (which align) and single stranded U-runs

28
Q

extrinsic/factor mediated termination

A

rho termination

recognition site (rut site) and effect site of rho are different

29
Q

summary termination

A

1.transient pausing–backtracking, hairpins, misincorporation
(there is transient pausing which does not lead to termination)

  1. in such cases RNA can be cleaved by polymerase to give new free 3‘ -OH = backtracking
  2. termination: intrinsic(e.g. hairpin) or extrinsic(rho factor)
30
Q

polycistroniic transcript? definition?

A

in procaryotes!

=a transcript which carries several ORFs

an open reading frame goes from a start codon to a stop codon

in procaryotes, one nascent polycistronic mRNA (which extend from the DNA) can be covered by several ribosomes at the same time as it contains sweveral ORFs and several polypeptides can be made from one mRNA strand

31
Q

differences of transcription in pro- and eukaryotes

A
  • eu: Chromatin (Histones * DNA), several linear chromosomes
    pro: naked DNA, one circular chromosome
  • eu: RNA Pol needs GTFs for initiation
    pro: holoenzyme (= RNA Pol + sigma factor) binds directly
  • eu: 3 polymerases:
    pro: only one
  • eu: processing of primary transcripts to mRNA, introns
    pro: no processing??
  • eu: monocistronic mRNA (only?)
    pro: polycistronic mRNA, operons
  • eu: majority of DNA is non coding sequences
    pro: few

-eu: mRNA editing (5’ cap and 3’ poly A)

32
Q

which group of organisms has several RNA polymerases? name it!

A

eukaryotes

  • RNA pol I: 18S/28S rRNA
  • RNA pol II: mRNA (and few small RNAs)
  • RNA pol III: tRNA, 5S rRNA, other small RNAs
33
Q

typical regulatory seq of a Pol II transcribed gene

A

promoter

enhancer

silencer

34
Q

features of an enhancer/silencer

A

-binds transcriptional regulators
-increases promoter utilization
-can be upstream, inside the gene or downstream
(„distal“ enhancers can be veryfar away)
-orientation not important
-often target for tissue-specific or temporal regulation

35
Q

how can distal enhancers/silencers influence?

A

DNA loops bring enhancers and promoters together

36
Q

study of chromatin looping

A

3C technique

3C“ = Chromosome Conformation Capture

37
Q

The mediator complex

A

The mediator complex links transcriptional regulators with the basal transcription apparatus

38
Q

Integration at promoters

A

Promoters can function as genetic switches that integrate regulatory information
MODULARITYof regulatory input is a recurring theme

39
Q

Enhancer:

A

activating regulatory sequence separate from core promoter
–independent from distance and orientation

2.Enhancers bind activating transcription regulators(repressingfactors bind to silencers)

40
Q

Enhancers may function

–in

A

-making the promoter accessible
(chromatin remodelling and modifications)
–changing DNA topology (e.g.bending)
–interacting with the basal transcription apparatus

41
Q

eukarytic promoter

A

Modular: can contain e.g.

  • Inr–Initiator region
  • TATA box
  • DPE–Downstream Promoter Element (in TATA-less promoters)

CpG islands

Promoter recognition complexes bind the promoters
5.Classic example: TFIID(TBP and TAFs)

42
Q

instead of sigma?

A

in eu no sigma –role fulfilled by the GTFs
enzyme alone can transcribe, but not initiate

but: especially catalytic units conserved

43
Q

transcription factors

A

TFIIA: -TFDII can bind to region extending farther upstream
TFIIB: -binds adjacent to TBP (BRE -BRecognition Element)
-determines promoter polarity
-recruits the polymerase
TFIIF: -binds polymerase
-facilitates recruitment
and participates in DNA repair!

Open complex formation andpromoter clearance:

TFIIE: -facilitates formation of initiation-competent polymerase
-recruits TFIIH
TFIIH: -multiple enzymatic activities
-helicase -> melting of the DNA

44
Q

CTD code

A

Phosphorylation of the CTD regulates transcription and RNA processing

a ctd code for all different stages of transcription

e.g. promotor clearance

45
Q

TFIIH:

A

The TFIIH helicase function assists in promoter melting
3.The TFIIH kinase function phosphorylates the CTDdomain of the polymerase (Ser5) –promoter escape

participates in DNA repair

46
Q

PTEFbkinase

A

PTEFbkinase phosphorylates the CTD (Ser2) –productive elongation

47
Q

Transcription and RNA processing are integrated

A

Transcription and RNA processing are integrated –mediated by CTD code

48
Q

Transcription factories

A

Transcription factories -spatial organization of transcription in the nucleus

49
Q

Transcription and DNA repairare linked

A

Transcription and DNA repairare linked (TFIIH)

50
Q

what is required for promoter recruitment in eikaryotes?

A

TBP

Genetics (10 decks)

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