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Transcription Flashcards

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1
Q

Commonalities between Replication and Transcription

A
  1. same fundamental chemical mechanism
  2. same directionality of growth
  3. same use of DNA template
  4. same 3 phases
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2
Q

Differences between Replication and Transcription

A
  1. primer not used in transcription
  2. not all DNA transcribed
  3. one stranded of DNA template transcribed
  4. RNA polymerase has sloppy proofreading ability
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3
Q

RNA Polymerase

A
  • one type in prokaryotes
  • three types in eukaryotes
  • structure highly conserved
  • require DNA, ribonucleotides, magnesium ions for activity
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4
Q

E. Coli RNAP

A
  • 5 subunits: a, B, B’, w, sigma (two alpha subunits in actual composition)
  • holoenzyme contains sigma unit
  • sigma helps enzyme recognise specific promoters and initiate transcription before leaving
  • leaves core enzyme which carries out catalysis and elongation
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5
Q

Initiation

A
  • RDS is commitment to initiate and this is highly regulated
  • holoenzyme binds to 70bp before transcription start site
  • key conserved sequences occur at -35 and -10 regions
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6
Q

DNA footprinting experiment **

A
  • if a protein binds to a specific DNA sequence this protects DNA from nuclease cuts
  • this showed the critical binding sites for transcription enzymes
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7
Q

E. Coli Promoters

A

-35 region
-10 region
UP element
Start of transcription (usually A)

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8
Q

Sigma Factor Role

A

Decreases ability of core enzyme to bind DNA nonspecifically (DNAP has innate affinity for DNA so can bind randomly)

  • allows holoenzyme to bind promoters
  • allows holoenzyme to migrate along DNA until promoter encountered
  • different sigma factors permit binding to different promoters
  • allows specific regulated gene expression
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9
Q

Roles of Polymerase Subunits

A 
a : binding of regulatory/promoter sequences (in major groove)
B : forms phosphodiester bonds
B' : binds DNA template
*B/B' regions are the catalytic site*
sigma: promoter recognition
w : RNAP assembly and stability
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10
Q

Transcription Initiation/Binding

A
  • series of conformational changes
    1. closed complex forms at promoter
    2. open complex formation (partially unwound DNA)
    3. initiation (add first ribonucleotides)
    4. promoter clearance (loss of sigma factor and change in holoenzyme to only core enzyme)
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11
Q

Scrunching

A

Sigma doesn’t allow RNAP to move and mRNA accumulates

- either promotor dissociates and elongation occurs or transcription aborted

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12
Q

Elongation

A
  1. open complex between DNA and RNA unwinds 17bp at start site (transcription bubble)
  2. after promoter clearance, RNA chain is extended and the polymerase moves along DNA
  3. template DNA selects appropriate ribonucleotides by Watson Crick base pairing
  4. transcription bubble: 8 bp of DNA base paired to synthesized A form RNA
  5. Bubble moves at 50 nucleotides per second
  6. structure of RNA polymerase forces its exit from helix
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13
Q

Termination

A

1: requires rho (p) factor
2: doesn’t require rho factor

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14
Q

Rho dependent Termination

A
  • binds CA rich sequences of RNA

- uses ATP helicase activity to move up the mRNA and blow the RNAP off, causing mRNA release

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15
Q

Rho independent Termination

A
  • hairpin structure in RNA followed by UUU causes RNA pause and subsequent mRNA release
  • A-U pair weak
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16
Q

Eukaryotic Polymerases

A

Pol I: makes pre ribosomal RNA processed into 3 of the 4 types of rrRNA

Pol II: makes mRNAs and microRNAs

Pol III: makes tRNA,microRNA, 5S RNA

17
Q

a - aminitin

A
  • incredibly high binding affinity for polymerase II, inactivating it
18
Q

Eukaryotic Promoters

A
  • much more complex

Pol II promoters: adjacent conserved sequences necessary for formation of initiation complex but are more complex

  • TATA box and -30 and Inr sequence at +1
  • upstream element
19
Q

Eukaryotic Initiation

A
  • Pol II cannot initiate independently
  • requires assembly of large transcription complex
  • TFIID binds to TATA box
  • transcription factors 2A/2B
  • RNAP II chaperoned by TFIIF binds to form initiation complex (TFIIE,J,K)
  • TFIIH is kinase phosphorylating carboxyl terminus tail of Pol II - haploid repeats with Serine residues
  • Phosphorylation converts RNAP to elongation form to begin transcription
20
Q

Eukaryotic mRNA Modification

A
  • base modification
  • deletion/additions to ends
  • removal of introns in primary transcript
  • methyl guanosine cap on 5’ end
  • polyadenyl tail on 3’ end
  • introns removed by splicing
21
Q

DNA supercoiling

A

Negative supercoiling of circular DNA favours the transcription of genes because it facilitates
unwinding. The introduction of negative supercoils into DNA by topoisomerase II can
increase the efficiency of promoters located at distant sites

Molecular Biology (11 decks)

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