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Eukaryotic Gene Expression > L1 - Basal Transcription Machinery > Flashcards

L1 - Basal Transcription Machinery Flashcards

1
Q

What are the features of nuclear RNAPI?

A

14 subunits

Transcribes rDNA only

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2
Q

What are the features of nuclear RNAPII?

A

12 subunits

Transcribes most nuclear genes and most snRNA and miRNA

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3
Q

What are the features of nuclear RNAPIII?

A

17 subunits

Transcribes a few short genes e.g. tRNA genes, 5S rRNA, U6 snRNA

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4
Q

What are are the types of non-coding genes/

A

rRNA
tRNA
snRNA
miRNA

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5
Q

How many protein encoding and non coding genes do humans have?

A

23000 protein

4000 non coding

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6
Q

What is similar about bacterial rnap and eukaryotic rnap?

A

Share similar cores

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7
Q

What can mutations in Pol3 subunits cause?

A

Hypomyelinating neurodegenerative disorders

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8
Q

How is it thought mutations in pol3 cause neurodegenerative disorders?

A

Maybe certain CNS cells are especially sensitive to abnormal pol III activity or pol iii products only needed in certain CNS cells

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9
Q

What is TBP?

A

TATA Binding Protein

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10
Q

What does TBP do?

A

Binds TATA sequence in promoters
Bends DNA 90 degrees
Used by all 3 pols
Provides platform for binding of other TFs

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11
Q

How is the pol ii preinitiation complex formed?

A
  • TBP bends DNA to allow TFIIB to contact TFIIB recognition element (BRE) flanking TATA box
  • TFIIB attracts pol ii and TFIIF
  • TFIIE joins and recruits H
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12
Q

What are the features of TFIIH?

A 
10 subunits (XPB, XPD,Cdk7)
Dissociable kinase domain called CAK with a Cdk7 which phosphorylates CTD of pol ii
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13
Q

What can mutations in XPB or XPD subunits of TFIIH cause and why?

A 
Xeroderma pigmentosum (defective repair of UV damage, extreme photosensitivity)
Because TFIIH involved in DNA repair too
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14
Q

What does XPB helicase do?

A

Melts DNA and feeds template into pol ii

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15
Q

What does TFIIB do?

A

Stabilises open DNA and recognises TXN start

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16
Q

What is the transcription bubble and what are its features?

A

8-9 nts of RNA remain annealed to DNA template, as 3’ RNA extends, 5’ peels away from DNA
Melting at front of bubble balanced with reannealling at back so size constant

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17
Q

What are the key features of the active site of pol ii?

A

Template nt forms Watson crick base pairing with incoming NTP
3’ end of RNA stacks on base of incoming NTP
Mobile trigger loop forms hairpin when NTP binds

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18
Q

How is the phosphodiester bond formed?

A

NTP condensation cataltsed by two Mg2+ residues
MgA promotes deprotonation of 3’ RNA OH group
Resultant O group attacks substrate NTP phosphate
MgB stabilises transition state
Bond forms, PPi released with MgB

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19
Q

What speed can pol ii elongate at and with what help?

A

2000 per minute with elongation factors

20
Q

What does TFIIS do?

A

allows arrested polii to resume txn when it has disengages

21
Q

How does TFIIS unarrest txn?

A

Zinz ribbon inserts into active site to stimulate nuclease activity to cleave RNA, creating new RNA 3’ end that is correctly positioned for txn

22
Q

What is RPB1 and what are its features?

A

Largest subunit of pol ii
Has a CTD which is of 7 residue repeats
Number of repeats varies with species, humans have 52

23
Q

What does RPB1 do?

A

Provides docking platform for proteins according to its phos state

24
Q

Why does the phosphorylation state of RPB1 matter?

A

Varies through txn cycle allowing dynamic recruitment of proteins

25
Q

What happens during the dynamic phosphorylation cycle of pol ii?

A

Recruited unphos
H phos Ser5 and 7 when txn initiates
During elongation Ser5 gradual unphos and Ser2 gradual phos

26
Q

What function does the dynamic phosphorylation cycle give?

A

CTD code shows where polii is along a gene

27
Q

How is the CTD code read and used?

A

Mediator binds:

  • unphos CTD - brings mRNA capping enzymes
  • Ser5p and Ser2p recruits elongation and splicing factors
  • Low Ser5p and high Ser2p recruits processing and export proteins
28
Q

What do most mRNAs end in?

A

AAUAAA

29
Q

What does CPSF do?

A

Recognises end of mRNA, cleaves and adds polyA tail

30
Q

What does the poly A tai ldo?

A

Enhances export, stability and translation

31
Q

What happens after poly adenylation?

A

Exonuclease attacks unprotected 5’ end of RNA still attached to poly ii and then DNA released from DNA

32
Q

What does pol I do?

A

Transcribes single rDNA gene to make rRNA precursor

33
Q

How is pre-rRNA processed?

A

28S, 18S and 5.8S rRNAs

34
Q

How much of a cells RNA is rRNA?

A

80%

35
Q

What are NORs?

A

Nucleolar organiser regions

200 copies of rDNA in tandem repeats clustered on five human chromosomes

36
Q

What happens in cancer and how can it be diagnosed?

A

Synthesis of rRNA elevated and nucleoli form around it.

Increased number and morphology of nucleoli indicator of tumourigenesis

37
Q

What factors contribute to the prognosis of cancer?>

A

Smaller nucleoli and slower growth = better prognosis

38
Q

How is Pol 1 txn initiated?

A

UBF is TF
SL1 is tf found at promotor
Complex of pol 1 with TIF-IA recruited by SL1

39
Q

What occurs in pol I elongation and termination?

A

Stops at termination sites boun dto TTF-1

PRTF assists release

40
Q

What are the TFs for pol iii and what are their functions?

A

C binds promoter
C recruits B
iii recruited by B

41
Q

Why does pol iii have no termination factors?

A

Can return to start with out dissociating

42
Q

What are the similarities of the pols?

A

TBP is part of SL1 and TFIIIB

Several subunits found in pol1 and 3 correspond to TFs of pol ii

43
Q

What is interesting about the evolution of pols 1 and 3?

A

Involved attachment of txn factors that interact more loosely with pol 2

44
Q

How is each pol inhibited?

A

a-amanitin toxin
1 is resistant
2 is inhibited by low doses
3 is inhibited by intermediate doses

45
Q

What controls are used when using toxin to inhibit pols?

A

GAPDH as + control to confirm treatment efficacy

tRNAtyr as - as pol III is les sensitive to toxin than 2

Eukaryotic Gene Expression (8 decks)

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