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Genes and Bioinformatics > Week 4a - Mechanisms of Eukaryotic Transcription > Flashcards

Week 4a - Mechanisms of Eukaryotic Transcription Flashcards

1
Q

Prokaryotes and bacteriophages

A

no nucleus

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2
Q

Eukaryotes

A

• nucleus
• enzymes must get in (pores in nuclear envelope)
• more
genes, more complex

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3
Q

Introns are removed at the level of

A

mNRA splicing

• 1 gene –> more than 1 type of RNA (alternative splicing)

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4
Q

Eukaryotes vs prokaryotes

A
  • eukaryotes have introns and exons

* prokaryotes have operon

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5
Q

Interrupted gene

A

a gene in which the coding sequence is not continuous due to the presence of introns

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6
Q

Exon

A

a segment of DNA that is transcribed and retained in the mature RNA product

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7
Q

Intron

A

a segment of DNA that is transcribed, but later removed from within the transcript by splicing together the sequences (exons) on either sides of it

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8
Q

Mature transcript

A

a modified RNA transcript

• modification may include removal of intron sequences and alterations to the 5’ and 3’ ends

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9
Q

Eukaryotic RNA polymerases

A

• eukaryotes have 3 RNA polymerases with distinct properties and one each specifically dedicated to rRNA, mRNA, and tRNA synthesis

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10
Q

Eukaryotic RNA polymerases require

A

numerous transcription factors

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11
Q

Eukaryotic RNA polymerase requires factors to

A

modify chromatin structure to allow RNA polymerase access

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12
Q

Eukaryotic RNA polymerase I

A

• located in the nucleolus
• makes pre-rRNA
(leading to 5.8s, 1BS, and 28S rRNA)
• 14 subunits

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13
Q

Eukaryotic RNA polymerase II

A
  • located in the nucleoplasm
  • makes pre-mRNA and some snRNAs
  • 12 subunits
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14
Q

Eukaryotic RNA polymerase III

A
  • located in the nucleoplasm
  • tRNA, 5srRNA, U6 snRNA (spliceosome)
  • 7SL RNA (signal recognition particle)
  • 17 subunits
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15
Q

Eukaryotic RNA polymerases share some subunits but not others

A 
in RNA polymerase I, II, III
• Rpb5
• Rpb6
• Rpb8
• Rpb10
• Rpb12
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16
Q

Many RNA polymerase II subunits

A

have homologs in RNA polymerase I and/or III

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17
Q

Bacterial RNA subunits have

A

at least 1 homolog in each eukaryotic polymerase

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18
Q

Structure of RNA pol II

A
  • 2 catalytic centers

* magnesium ion in active site

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19
Q

Trarnscription initiation can be

A

focused or dispersed
• RNA polymerase can’t initiate transcription from specific start sites without the assistance of other proteins - transcription factors
• each RNA polymerase has its own specific set of transcription factors to assist in locating their specific start sites
• around each transcription site is a core promoter
• approximately 70% of vertebrate core promoters are dispersed promoters (allows multiple mRNAs to be produced from a single gene)

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20
Q

RNA polymerase cant initiate transcription from specific start sties without

A

the assistance of other proteins - transcription factors

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21
Q

Each RNA polymerase has its own specific

A

set of transcription factors to assist in locating their specific start sties

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22
Q

Around each transcription site is a

A

core promoter

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23
Q

Approximately 70% of vertebrate core promoters are

A 
dispersed promoters
• allow multiple mRNAs to be produced from a single gene
• often by cell type, tissue specific 
(different place = different promoter)
- by different transcription factors
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24
Q

More promoters mean

A

transcription at different spots
–> more than 1 mRNA
• more than 1 transcriptional start site
–> diversity in RNA

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25
Q

Eukaryotic core Pol II promoter motifs

A
  • TATA box: TATAXAX consensus sequence (X is A or T) located 25-30 bp upstream of the transcription start site
  • initiator element (lnr) - overlaps the transcription initiation site (binds TFIID)
  • downstream promoter element (DPE) - extends from about +28 to +34
  • TFIIB recognition element (BRE) - accessory transcription factor
  • SCPE1 - present in TATA-less human core promoters (1% human genes)
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26
Q

TATA box

A

TATAXAX consensus located 25-30 bp upstream of the trascription start site
(X is A or T)

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27
Q

Initiator element (Inr)

A

overlaps the transcription initiation site

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28
Q

Downstream promoter element (DPE)

A

extends from about +28 to +34

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29
Q

TFIIB recognition element (BRE)

A

accessory transcription factor

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30
Q

SCPE1

A

present in TATA-less human core promoters

1% human genes

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31
Q

RNA polymerase II requires several

A

general transcription factors

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32
Q

TFIIA

A

general transcription factor for RNA polymerase II
• 2 subunits
• stabilizes TBP and TFIID binding
• blocks the inhibitory effects of TAF1 and other proteins

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33
Q

TFIIB

A

general transcription factor for RNA polymerase II
• 1 subunit
• stabilizes TFIID-promoter binding
• contributes to transcription start site selection
• helps recruit RNA polymerase II TFIIF to the core promoter

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34
Q

TFIID

TBP and TAFs

A

general transcription factor for RNA polymerase II
• 1 and 14 subunits
• binds TATA element and deforms promoter DNA
• platform for the assembly of TFIIB and TAFs
• binds Inr, MTE, DPE, and DCE promoter elements

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35
Q

TFIIE

A

general transcription factor for RNA polymerase II

• helps to recruit TFIIH to the core promoter and is required for promoter melting

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36
Q

TFIIF

A

general transcription factor for RNA polymerase II
• 3 subunits
• binds RNA polymerase II
• involved in recruiting the polymerase to the pre-initiation complex
• required to recruit EFIIE and EFIIH to the pre-initiation complex

37
Q

TFIIH

A

general transcription factor for RNA polymerase II
• functions in transcription and DNA repair
• it has kinase and helicase activities
• is essential for open complex formation

38
Q

Picture from slide 10

A

*

39
Q

Pre initiation complex assembly (onto TATA box)

A
  • accessible core promoter
  • TFIID (core promoter recognition)
  • TFIIA
  • TFIIB
  • TFIIF - Pol II
  • TFIIE
  • TFIIH
40
Q

TFIID has

A

TATA binding protein (TBP)

41
Q

TFIIA stabilizes

A

to let TFIIB bind (also stabilizes and promoter escape, results TFIIF)
• RNA polymerase binds after 3rd step, doesn’t bind TATA box

42
Q

TBP is the TATA binding subunit of

A

TFIID
• TFIID can bind to the promoter without the assistance of other proteins
• TBP is saddle shaped and bends DNA by 80degrees
• interaction between TBP and the TATA box involves conformational changes in both TBP and the TATA box
• TBP interacts with TATA in the minor groove

43
Q

TFIID can bind to the promoter

A

without the assistance of other proteins

44
Q

TBP is saddle shape and bends the DNA by

A

80 degrees

45
Q

Interaction between TBP and the TATA box involves conformational changes in

A

both TBP and the TATA box

46
Q

TBP interacts with the TATA box in

A

the minor groove

47
Q

TBP-associated factors (TAFs) bind

A

promoter elements other than TATA
• additional proteins present in TFIID are called TBP-associated factors
• TFIID contains a core set of 13 TAFs
• TAFs are required for high levels of transcription and to transcribe genes that lack a TATA box
• TATA-less promoters - it is the TAFs that bind to regulatory elements

48
Q

Additional proteins present in TFIID are called

A

TBP-associated factors (TAFs)

49
Q

TFIID contains a core set of

A

13 TAFs

50
Q

TAFs are required for

A

high levels of transcription and to transcribe genes that lack a TATA box
• anchor TFIID to promoter (additional contact with promoter)

51
Q

In TATA-less promoters -

A

it is the TAFs that bind to regulatory elements

52
Q

The transcription initiation process

A
  1. TBP binds the TATA box around the C-terminal domain of TFIIB (DNA bent around TFIIB)
  2. the N-terminal domain of TFIIB brings the complex to RNA polymerase II and positions the transcription initiation site in the active site of RNA polymerase
  3. The RNA polymerase II-TFIIB-promoter complex recruits TFIIE which then recruits TFIIH
  4. the helicase activity of TFIIH unwinds DNA in the vicinity of the initiation site
  5. TFIIF captures the non-template strand after unwinding
  6. the template strand descends to the active site
53
Q

The major transitions from transcription initiation to elongation

A
  1. open complex formation - initiation of transcriptional bubble
  2. RNA chain initiation - first 2 ribonucleotides line up on template strand in the transcription bubble and RNA polymerase catalyzes phosphodiester bond formation
  3. abortive transcription - formation of short transcripts which are released
  4. promoter escape - chain extension beyond 7 ribonucleotides triggers TFIIB release and formation of the transcriptional elongation complex
54
Q

All parts are kept

A

separate in the catalyitic core

55
Q

Polymerase must escape

A

being held at promoter

56
Q

RNA polymerase must be

A

phosphorylated for elongation

57
Q

RNA polymerase must be phosphorylated for elongation

A
  • carboxyl terminal domain (CTD) of the largest subunit in RNA polymerase II (Rpb1) has important role in transition from the initiation complex to the elongation complex
  • TFIIH has 10 subunits, is ring shaped, and has 5’–>3’ and 3’–>5’ helicase activies
  • also has cyclin-dependent protein kinase activity
  • Ser-5 phosphorylation of RNA pol II permits promoter clearance
  • Ser-2 and Ser-7 are also phosphorylated in RNA pol II during elongation
  • dephosphorylated after each round of transcription before RNA pol II can form another PIC
58
Q

Modification of RNA pol II CTD

A
  1. recruitment of pol II by the pre initiation complex
  2. phosphorylation on Ser5 by TFIIH helps recruit enzymes to cap the 5’ end of the transcript
  3. phosphorylation on Ser2 by P-TEFb activates elongation, splicing, and polyadenylation
  4. elongation of pol II
  5. phosphorylation on Ser7 by an unknown kinase has so far no known role in expression of protein-coding genes
  6. Elongating pol II splicing
  7. dephosphorylation by Ser5 phsophatase
  8. binding of polyadenylation factors to Ser2 phosphorylated repeats with peptidyl-prolyl bonds in the trans configuration
  9. cleavage, polyadenylation, and termination
  10. dephosphorylation by Ser2 phosphatase (eg Fcp1), dephosphorylation by Ser7 phosphatase?
  11. glycosylation? (phsophorylation by Cdk8/Srb10, ERK1/2, CDC2/CDK1)
59
Q

Modification of RNA pol II CTD - simple

A
  • unphosphorylated RNA polymerase recruited by THIIF
  • phosphorylated on 2nd serine escapes from promoter
  • phosphorylate serine 5 and 7
  • dephosphorylate, release unphosphorylated mRNA
60
Q

The transcription elongation complex

A
  • more stable than the initiation complex
  • approximately 14bp are melted to form the transcription bubble
  • first 8 nucleotides within bubble are paired with RNA chain
  • double stranded DNA opens up in front of the bubble and closes up behind the bubble as RNA polymerase moves along
  • the transcription bubble extends from -12 to +2
  • phosphorylation of CTD is involved in processing mRNA transcript
  • phosphorylation of CTD provides binding/recognition sites for mRNA processing
61
Q

The transcription elongation complex is more stable than

A

the initiation complex

62
Q

For the transcription elongation complex aproximately 14 bp are melted to form

A

the transcription bubble

63
Q

In the transcription elongation complex the first 8 nucleotides are paired with

A

the RNA chain

64
Q

In the transcription elongation process, double stranded DNA

A

opens up in front of the bubble and closes up behind the bubble as RNA polymerase moves along

65
Q

The transcription bubble extends

A

from -12

to +2

66
Q

In the transcription elongation complex phosphorylation of CTD is involved in

A

processing mRNA transcript

67
Q

In the transcription elongation complex phosphorylation of CTD provides

A

binding/recognition sites for mRNA processing

68
Q

RNA polymerase does not move

A

at a steady pace
• once elongation starts, RNA polymerase remains associated
• pauses a lot because RNA polymerase puts on wrong base, so stops and backs up
• repaired by TFIIS (not needed for initiation but for elongation)

  • chain elongation is temporarily delayed at pause sites
  • pausing may lead to arrest and termination
  • arrest is an important step in proofreading
  • TFIIS reactivates arrested RNA polymerase
69
Q

Chain elongation is temporarily

A

delayed

at pause sites

70
Q

Pausing may lead to

A

arrest and termination

71
Q

Arrest is an important step

A

in proofreading

72
Q

TFIIS

A

reactivates arrested RNA polymerase

73
Q

RNA polymerase I has a

A

bipartite promoter

74
Q

RNA polymerase i has a bipartite promoter

A
  • RNA polymerase I promoters consist of a core promoter and an upstream promoter element (UPE)
  • the factor UBF1 wraps DNA around a protein structure to bring the core and UPE into proximity
  • SL1 includes the factor TATA-BINDING PROTEIN that is involved in initiation by all 3 RNA polymerases
  • RNA polymerase I binds to the UBF1-SL1 complex at the core promoter
75
Q

RNA polymerase I consists of

A
  • a core promoter

* an upstream promoter element (UPE)

76
Q

The factor UBF1

A

wraps DNA around a protein structure to bring the core and UPE into proximity

77
Q

SL1 includes the factor

A

TATA-binding protein that is involved in initiation by all 3 RNA polymerases

78
Q

RNA polymerase I binds

A

to the UBF1-SL1 complex at the core promoter

79
Q

RNA polymerase III uses

A

downstream and upstream promoters

80
Q

RNA polymerase III uses downstream and upstream promoters

A
  • RNA polymerase III has 3 types of promoters, 2 types of internal promoters
  • internal promoters have short consensus sequences located within the transcription unit and cause initiation to occur at a fixed distance upstream
  • upstream promoters contain 3 short consensus sequences upstream of the start point that are bound by transcription factors
  • TFIIIA and TFIIIC bind to the consensus sequences and enable TFIIIB to bind at the startpoint
  • TFIIIB has TBP as one subunit and enables RNA polymerase to bind
  • preinitiation complex formation
81
Q

RNA polymerase III has

A

3 types of promoters

2 types of internal promoters

82
Q

Internal promoters have

A

short consensus sequences located within the transcription unit and cause initiation to occur at a fixed distance upstream

83
Q

Upstream promoters contain

A

3 short consensus sequences upstream of the start point that are bound by transcription factors

84
Q

PSE=

A

proximal sequence element

85
Q

TFIIIA and TFIIIC bind to

A

the consensus sequences and enable TFIIIB to bind at the start point

86
Q

TFIIIA and TFIIIC bind to the consensus sequences and enable

A

TFIIIB to bind at this start point

87
Q

TFIIIB has TBP as one subunit and enables

A

RNA polymerase to bind

88
Q

boxA and boxB bind

A

TFIIIC

Genes and Bioinformatics (10 decks)

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