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Biochemistry > Transcription-general principles > Flashcards

Transcription-general principles Flashcards

1
Q

The mRNA sequence is the same as the…

A

Non-template strand/+ strand

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2
Q

What are the features of prokaryotic RNApol

A

One RNA pol that transcribes all RNAs

Core enzyme has 4 subunits – α2ββ’ – these catalyze RNA synthesis
ω subunit binds core and facilitates holoenzyme formation
σ factor binds core – σ factor directs polymerase to the promoter

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3
Q

What are the different prokaryotic sigma factors?

A

There are multiple σ factors in E. coli

  • σ70 responsible for binding to most promoters in E. coli
  • σ32 responsible for binding promoters of heat shock genes
  • σ28 responsible for binding flagellin gene promoter
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4
Q

How does transcription begin?

A

RNApol binds to promoter

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5
Q

How is promoter strength determined?

A

Strong promoters are used frequently,
weak promoters are used less frequently. Strength is dictated by sequences at -35
and the Pribnow box

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6
Q

What is the -35 sequence?

A

sequence in the promoter 35 nts before start site

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7
Q

What is the Pribnow box?

A

– Pribnow box – occurs at -10 (i.e. 10 bp before the initiation site)

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8
Q

Where is the promoter located?

A

5’ end of the gene

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9
Q

What happens when RNApol holoenzyme binds to promoter?

A

Holoenzyme binds to promoter
- σ factor or β’ unwinds DNA at Pribnow sequence
- enzyme/promoter complex is now called “open”
Ribonucleotides can now base pair with the template strand

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10
Q

How does RNApol work?

A

RNA Pol catalyzes polymerization
- like DNA synthesis – mechanism is nucleophillic attack by 3’-OH
group on the α-phosphate on the following nucleotide

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11
Q

What is located at the 5’ end of a prokaryotic mRNA?

A

triphosphate

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12
Q

What must happen for transcription to proceed into the elongation phase?

A

RNA Pol must successfully synthesize an RNA ~10 nts long in order to
progress to the next phase
- if it fails – the little RNA is release and degraded – Pol tries again
- if it succeeds – conformation of Pol changes, σ factor is released
and proceeds to elongation

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13
Q

What is a transcription bubble?

A

Transcription bubble – segment of DNA that is temporarily single stranded during
transcription – this is 12-14 nts long
Note that RNA Pol covers ~30 nts – therefore transcription bubble protected
by RNA Pol

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14
Q

How is ssDNA protected during transcription?

A

Blocked by RNA pol

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15
Q

What is the channel structure of RNA polymerase?

A

RNA Pol has 3 channels – coding strand passes through one channel
- template strand passes through second channel – nucleotides bind
here and synthesis occurs
- RNA passes through a third channel to be released
The channels containing the coding and template strands end in proximity so the two strands can rapidly re-anneal

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16
Q

What enzyme relieves supercoiling tension during transcription?

A

topoisomerases

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17
Q

How does intrinsic termination work?

A

Intrinsic termination – termination sequence has an inverted repeat
followed by a string of U’s (in the RNA)
- this repeat can form a strong, stable hairpin
- hairpin stalls the RNA Pol
- U’s in the RNA base paired to A’s in the template strand are
weak
- RNA is released, transcription terminates

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18
Q

What is an inverted repeat?

A

5’ -> 3’ sequence on the template strand is the same as a 5- -> 3’ sequence on the non-template strand further downstream

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19
Q

How does rho-dependent termination work?

A 
Rho factor – ATP-dependent helicase
- binds a specific sequence in
nascent RNA
- translocates along RNA
- unwinds DNA/RNA duplex
- dislodges RNA Pol and
transcription terminated

Believed to be a weak hairpin formed
downstream of rho binding site to stall
RNA Pol

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20
Q

How can histones be modified? What are the effects on transcription?

A

Histones can be posttranslationally modified, e.g. acetylation, methylation, phosphorylation
- modifies histone interactions to alter chromatin structure
- e.g. need histone acetylation for access to promoters
histone acetyl transferases (HATs) – promote gene expression
histone deacetylases (HDACs) – impair gene expression

21
Q

How can protein complexes modulate histone interactions?

A
  • SWI/SNF complexes promote histone release from DNA

- NURF proteins weaken histone/DNA interactions so they can slide

22
Q

What RNAs do each of the 3 RNA polymerases in eukaryotes transcribe?

A

RNA Pol I – transcribes larger rRNAs
RNA Pol II – transcribes mRNA precursors, miRNAs and snRNAs
RNA Pol III – transcribes tRNA, 5S rRNA, U6 snRNA, snoRNAs

23
Q

How do eukaryotic RNA pols differ from prokaryotic?

A

Eukaryotic requires additional transcription factors

24
Q

What are the parts of eukaryotic promoters?

A

Three parts – core promoter – required for RNA Pol binding
- proximal promoter elements – transcription factors bind

  • distal promoter elements – can be upstream or downstream
  • function to enhance or repress transcription
  • binding sites for enhancers or repressors
25
Q

What are the two types of eukaryotic promoters?

A

focused promoter – has a major transcriptional
start site (TSS)
- dispersed promoter – multiple TSSs over 50-100 nts

26
Q

What is a focused promoter? What is an example?

A

Set of core promoter elements

examples of CPEs – TATA box

  • bound by TATA box binding protein (TBP)
  • part of TFIID
  • initiator (Inr)
  • downstream of promoter element (DPE)
27
Q

What are the characteristics of dispersed promoters?

A

Usually occur in CpG islands, and with housekeeping genes

28
Q

What are examples of proximal promoter elements?

A

Proximal promoter elements – within about 250 bp of TSS

  • examples – GC box – bound by transcription factor called SP1
  • CAAT box -
29
Q

Where can distal promoter elements be located? What are their functions?

A

Distal promoter elements – further away

  • upstream, downstream or even in introns
  • enhancers or repressors
30
Q

What is the major subunit of RNA Pol II

A

RBP1

31
Q

What are the features of RBP1?

A

RBP1 – has a C-terminal domain (CTD)

  • has a repeated sequence YSPTSPS
  • up to 52 repeats of the sequence
  • site of posttranslational modification – phosphorylation
32
Q

How does phosphorylation affect RBP1 activity?

A 
unphosphorylated – binds promoter
- Ser2 and Ser5 phosphorylation change to regulate
switch to processivity
histone remodelling
5’ cap formation
splicing initiation
33
Q

What 6 binding factors does RNA Pol 2 bind? Which is the major one?

A

TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH
- these facilitate assembly of pre-initiation complex

TFIID is major

34
Q

What is the mediator complex?

A

Mediator - ~25 subunit protein complex
- binds to RNA Pol II and binds to enhancers and repressors bound to
distal promoter elements

35
Q

What is the function of TFIIH factor?

A

TFIIH – helicase – ATP-dependent unwinding of DNA to make transcription bubble

36
Q

What does RNA Pol II need to do to clear the promoter and proceed to elongation?

A

RNA Pol II needs to make 23 nts RNA for promoter clearance

  • TFIIE, TFIIH release for clearance
  • mediator release – go to elongation
37
Q

What is the transcription termination sequence? What happens when it is recognized?

A
  • AAUAAA
  • this is recognized by several proteins bound to the CTD
  • cleave transcript 10-30 nts downstream of this sequence
38
Q

How is the poly-A tail generated?

A

Poly(A) polymerase – generates a poly(A) sequence 100-250 As long
- covalently linked to the 3’ end of the message

39
Q

Are all mRNAs polyadenylated?

A

No

40
Q

What are hnRNPs?

A

Pre-mRNAs associate with 20 different types of proteins to form hnRNP
- heteronuclear ribonucleoprotein particles

41
Q

How is the 5’ end of pre-mRNA modified?

A

5’ end of pre-mRNA is modified – a 5’ cap is added
- 7 methyl guanosine is covalently linked
to the 5’ end via triphosphate linkage
- protection from nucleases
- ribosome recognition

42
Q

What catalyzes splicing?

A

The spliceosome–a large RNA/protein complex

43
Q

What sequences within the introns are recognized as splice sites?

A

GU-AG and AU-AC

44
Q

What is the branch site?

A

Another important nucleotide in the intron is an A near the 3’ end of the intron.
This is called the branch site

45
Q

What reactions occur at the branch site?

A 
Two reactions occur:
1) 2’-OH of branch site A
attacks the phosphate at the 5’
splice site – transesterification rxn
- intron forms a lariat

2) Lariat is cleaved – 3’ OH of upstream
exon attacks P next to lariat

46
Q

What is the result of the branch site reactions?

A

Exons are spliced together, intron is removed

47
Q

What is an exon junction complex?

A

Complex that binds to mRNA 22 nts upstream of each exon junction

48
Q

What is the function of EJCs?

A

Regulating nonsense mediated decay

If stop codons are found between EJCs, mRNA is degraded

Biochemistry (43 decks)

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