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M2M Unit 1 > Transcription > Flashcards

Transcription Flashcards

1
Q

How are phosphodiester bonds formed by RNA polymerase?

A

3’OH attacks alpha phosphate attached to 5’ carbon (releases 2 P groups)

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2
Q

5 steps in transcription cycle

Initiation, Elongation, Termination

A

1) RNA pol binds promoter sequence on helical DNA in a closed complex
2) Pol melts DNA strands apart near transcription start site forming an open complex
3) Polymerase catalyzes phosphodiester linkage of two initial rNTPs
4) Polymerase advances 3’–> 5’ down template strand, linking rNTPs 5’–>3’
5) At transcription STOP site, polymerase releases completed RNA and dissociates from DNA

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3
Q

RNA pol I

A

makes rRNA

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4
Q

RNA pol II

A
  • makes mRNA, snRNA, microRNA
  • CTD
  • Needs general transcription factors (TFIIH, TFIID)
  • Uses sliding clamp
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5
Q

C-terminal Domain (CTD)

A
  • on RNA pol II

- acts as a landing pad for proteins that effect elongation/processing

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6
Q

RNA pol III

A

makes tRNA

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7
Q

Promoter

A
  • sequence of DNA upstream of the transcription start site that direct RNA polymerase to the start of genes
  • controls FREQUENCY of transcription
  • site of pre-initiation complex assembly
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8
Q

TATA Box

A
  • promoter sequence (TATAAA)
  • 30 bases upstream from start site
  • conserved
  • site where GTFs bind
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9
Q

TATA box binding protein

A
  • binds TATA box

- directs assembly of pre-initiation complex of general transcription factors at the promoter

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10
Q

+1 base

-) and (+

A

-first base pair where transcription initiates

(-) = upstream
(+) = downstream
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11
Q

Proximal Promoter Elements

A
  • gene specific sequence that specific TFs bind

- Influence activity of the promoter

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12
Q

Enhancer

A
  • either up or down stream
  • can be very far away
  • bind transcriptional activator proteins and effect transcription
  • cell type specific
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13
Q

a-amanitin (and transcription)

A
  • non-competitive inhibitor of RNA polymerase

- Binds bridge helix of RNA pol II blocking translocation

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14
Q

Rifampicin (and transcription)

A
  • Inserts itself into RNA exit channel of bacterial RNA polymerase
  • Block growth of the RNA chain
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15
Q

TFIID

A
  • part of RNA polymerase II pre-initiation complex
  • aka TATA binding protein
  • promoter recognition GTF
  • binds TATA box on DNA sequence
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16
Q

TFIIH

A
  • part of RNA polymerase II pre-initiation complex
  • promoter recognition GTF
  • Functions in transcription and DNA repair (nucleotide excision)
  • has multiple subunits (CDK7 protein kinase and XPB helicase)
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17
Q

CDK7 Protein Kinase

A
  • part of TFIIH
  • phosphorylates RNA pol II CTD during promoter clearance
  • Allows pol II to elongate
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18
Q

XPB helicase

A
  • part of TFIIH

- acts as helicase to open up DNA strands

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19
Q

Syndromes associated with mutations in TFIIH (3)

A
  • Xeroderma Pigmentosum (XP)
  • Cockayne’s Syndrome (CS)
  • Trichothiodystrophy (TTD)
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20
Q

Xeroderma Pigmentosum (XP)

A

mutation in TFIIH so that is is unable to carry out nucleotide excision repair for damage caused by UV light

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21
Q

3 ways pre-mRNAs are processed

A
  1. Capping
  2. Splicing
  3. Cleavage/Polyadenylation
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22
Q

Capping

A

replacement of 5’triphosphate from the first rNTP with a backwards 7-methylguanosine (no phosphate group)

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23
Q

Splicing

A

excision of introns, desegmentation of exons

24
Q

Cleavage/Polyadenylation

A

cleavage of RNA at 3’end past consensus sequence, and polyadenylation (addition of > 200A’s) of cleaved site

25
Q

Pre-mRNA

A
  • initial product of transcription that must be extensively processed into a mature form (mRNA)
  • has introns AND exons
  • no cap, no tail
26
Q

Processing into mRNA enhances _________, _________, and ___________

A

nuclear export, translation, preventing degredation

27
Q

Function of 5’cap of mRNA (4)

A
  1. Protects the 5’end from exonucleases
  2. Recognized by cap-binding proteins - crucial for splicing/processing
  3. Recognized by TF eIF4E for nuclear export into cytoplasm
  4. Removal of cap signals mRNA degredation
28
Q

3 reactions required to add a 5’ cap to pre-mRNA

A

1) Triphosphatase - removes gamma phosphate at end of primary transcript
2) Guanylytransferase - synthesizes 5’–>5’ phosphodiester linkage
3) Guanine 7-methyltransferase - addition of methyl group at the 7 position

29
Q

Splice site at 5’ end of intron

A

GU

30
Q

Splice site at 3’ end of intron

A

AG

31
Q

Consensus sequence at poly-A site

A

AAUAAA

32
Q

Alternative splicing

A

allows many different proteins to be encoded by a single gene

33
Q

Genetic disorders caused by splicing defects (2)

A

1) CD44 - tumor metastasis

2) Spinal muscular atrophy

34
Q

CD44 and tumor metastasis

A

abnormal splicing of CD44 contributes to tumor metastasis

35
Q

Spinal muscular atrophy treatment

A

can activate alternate SMN2 gene to take over for defective SMN1 gene by using alternative splicing

36
Q

HIV and alternative splicing

A

HIV uses alternative splicing to generate many more mRNAs from a single transcript

37
Q

Steps of splicing (4) (trans-esterification reactions)

A

1) U2snRNA base pairs with branch point A, and activates it
2) U1 snRNA basepairs with 5’ splice site
3) 2’OH of branch point A then attacks phosphodiester bond IN FRONT of GU marker ath beginning of intron
4) 3’OH of exon 1 attacks phosphodiester bond at AG splice site, linking exon 1 and exon 2

38
Q

U2 snRNA

A
  • key role in splicing reactions

- base pairs with branch point A and activates it

39
Q

U1 snRNA

A
  • key role in splicing reactions

- base pairs with 5’ splice site

40
Q

Transcription start site

A

+1 position

41
Q

Introns

A

non-coding sequences between GU (5’) and AG (3’)

42
Q

branch point

A
  • single A residue
  • usually in intron between GU (5’) and AG (3’)
  • base pairs with U2 snRNA (VITAL part in first step of splicing)
43
Q

Exons

A

coding sequences

44
Q

5’UTR

A
  • untranslated region
  • between +1 and start codon on the processed mRNA strand
  • located at beginning of first exon
45
Q

3’UTR

A
  • untranslated region
  • located after stop codon, at the end of the last exon
  • contains highly conserved polyA site
46
Q

initiation/start codon

A
  • 5’AUG

- encodes methionine

47
Q

termination/stop codon

A

3’ UAG, UAA, UGA

48
Q

polyA site

A

consensus sequence AAUAAA

49
Q

Two reactions that make mature 3’ end of mRNAs

A

1) Cleavage: recognize consensus sequence (AAUAAA) at 3’ end of pre-mRNA and cleave
2) Polyadenylation: polyadenylation of free 3’ hydroxyl group

50
Q

3’ end processing of pre-mRNA is coupled with ________________ because ____________

A
  • termination of transcription by RNA pol II

- because 3’ polyA tail necessary for RNA polymerase complex to detach from the DNA

51
Q

Function of mRNA polyA tail (2)

A

1) protection from degradation

2) Export of mRNA from the nucleus

52
Q

Mutations in polyA site can result in _________

A

Thallasemia

53
Q

Thallasemia

A

results from mutations of the AAUAAA poly A site consensus sequence in globin genes

54
Q

IgM (immunoglobulin M) and alternative poly A sites

A

Membrane bound and secreted forms are formed based on alternative polyA sites in their common genome

55
Q

Cancer cells and alternative poly A sites

A

use altered polyA site choice to shorten the 3’UTR region, allowing them to escape regulation by microRNAs

M2M Unit 1 (9 decks)

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