Transcription

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Transcription

• Dr. Jason Linville
• University of Alabama at Birmingham
• jglinvil_at_uab.edu

from Genetics A Molecular Approach, 3rd ed.
Brown TA.
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Review

• Early experiments suggesting DNA is genetic
  material
• Structure of DNA by Watson and Crick
• Genes carry genetic information

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Summary

• Prokaryote vs. Eukaryote
• RNA Synthesis basics
• Transcription in E. coli
• Transcription in eukaryotes

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Prokaryote vs. Eukaryote
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Prokaryote vs. Eukaryote
PROKARYOTE
EUKARYOTE

• Lower organism
• Lack membranous organelles
• Bacteria and blue-green algae (cyanobacteria)

• Higher organism
• Have mitochondria, golgi, vesicles
• Unicellular and multicellular

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Prokaryote vs. Eukaryote

• Early genetic work done with prokaryotes
• Grew quick and easy in culture
• Eukaryotes not used successfully until 80s

• Gene expression is different in prokaryotes and
  eukaryotes, so relevance of work had to be
  accessed

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RNA Synthesis

• Template strand is read 3?5
• Ribonucleotides added in 5?3

• Addition of each ribonucleotide (triphosphate)
  results in loss of pyrophosphate molecule

• Addition of each ribonucleotide is base pair
  dependant (A?U, T?A, G?C)

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RNA Synthesis

• Beginning of transcription is not random.

• DNA-dependant RNA polymerase catalyses RNA
  formation reaction
• Just called RNA polymerase

• Where RNA polymerase attaches determines where
  transcription begins

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RNA Synthesis
RNA Polymerase structure varies between
prokaryotes and eukaryotes
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RNA Synthesis gt RNA Polymerase

• RNA Polymerase (prokaryote)

• In E.coli, there are 7000 RNA polymerase
  molecules (2000-5000 in use)

• RNA polymerase composed of 5 subunits
• a2ßßs holoenzyme
• a2ßß core enzyme

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RNA Synthesis gt RNA Polymerase

• RNA Polymerase (eukaryotes)

• Eukaryotes have 3 RNA polymerases
• RNA polymerase I, II, III
• Each transcribes a different set of genes
• Larger than prokaryotes 8-12 subunits
• 3 largest in yeast RNA polymerase II (RPB1, RPB2,
  RPB3) are similar to aßß in E.coli
• Polymerase can function in absence of some
  subunits

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RNA Synthesis gt RNA Polymerase

• RNA Polymerase (eukaryotes)

• RNA Polymerase I
• Ribosomal RNA

• RNA Polymerase II
• Genes for proteins most snRNA genes

• RNA Polymerase III
• Small RNAs, transfer RNA, 5S rRNA, other RNAs

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RNA Synthesis

• Initiation

• RNA polymerase binds upstream of gene

• Initiation site signaled by the promoter
• Promoter short nucleotide sequence recognized
  by RNA polymerase
• All promoters in E.coli have similar sequence
  (all recognized by same polymerase)

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RNA Synthesis gt Transcription gt E.Coli

• Initiation

• Promoter composed of 2 parts
• -35 box 5-TTGACA-3
• -10 box 5-TATAAT-3 (Pribnow box)

• Sequences of non-template polynucleotide
• Neg. number refers to the number of bases
  upstream from where transcription begins

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RNA Synthesis gt Transcription gt E.Coli

• Experiment

• DNase protection experiment
• See handout

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RNA Synthesis gt Transcription gt E.Coli

• Initiation

• Promoter composed of 2 parts
• -35 box 5-TTGACA-3
• -10 box 5-TATAAT-3 (Pribnow box)

• Some differences in promoter sequence appear to
  have no effect
• Some changes in sequence completely prevent RNA
  polymerase binding

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RNA Synthesis gt Transcription gt E.Coli
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RNA Synthesis gt Transcription gt E.Coli

• Initiation

• The s subunit of RNA polymerase recognizes
  promoter

• Therefore, the holoenzyme version of the RNA
  polymerase is required for recognizing promoter

• Initial structure is the closed promoter complex
  (holoenzyme attached to DNA)

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RNA Synthesis gt Transcription gt E.Coli

• Initiation

• After binding, the DNA base pairs break around
  the -10 region (melting) open promoter complex

• At this point the sigma subunit dissociates
  freeing the core enzyme to travel down the DNA

• As the sigma dissociates, two ribonucleotides are
  added

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RNA Synthesis gt Transcription gt E.Coli
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RNA Synthesis gt Transcription gt E.Coli

• Elongation

• The core enzyme travels along the DNA adding
  ribonucleotides that pair with the DNA template

• The transcript will be longer than the gene, with
  leader segments and trailer segments not coding
  for protein synthesis

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RNA Synthesis gt Transcription gt E.Coli

• Elongation

• Only a small amount of the DNA will be melted
  at one time. DNA hydrogen bonds reform after
  polymerase passes.

• Rate of elongation is not constant
• May even reverse and remove ribonucleotides
• Reasons unknown

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RNA Synthesis gt Transcription gt E.Coli

• Termination

• Not random. All termination is initiated by
  complementary palindrome.

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• Racecar
• Bar crab
• Flag Alf!
• Party-trap
• Gary knits a stinky rag.
• Kayak salad - Alaska yak.

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RNA Synthesis gt Transcription gt E.Coli

• Termination

• Not random. All termination is initiated by
  complementary palindrome.

• Two types
• not Rho-dependant
• Rho-dependant
• both still somewhat mysterious

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RNA Synthesis gt Transcription gt E.Coli

• not Rho-dependant

• After stem loop, run of As in template and run of
  Us in RNA are weak just breaks

Rho-dependant

• Rho large protein
• Attaches to growing RNA before stem loop
• Actively disrupts DNA/RNA pairing

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RNA Synthesis gt Transcription gt E.Coli

• Completion

• After RNA is free, core enzyme dissociates and
  reforms with sigma

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• Very similar to prok. more complex initiation

Initiation

• 3 polymerases 3 types of promoters

• Promoters more complex
• core promoter where polymerase binds
• surrounding sequences (hundreds of base pairs)
  can enhance or deter initiation

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RNA Synthesis gt Transcription gt Eukaryotes
Initiation

• RNA polymerase II forms pre-initiation complex
  with transcription factors (other proteins)

• Pre-initiation complex forms at
• -25 box 5-TATAAT-3 (TATA box)

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RNA Synthesis gt Transcription gt E.Coli
Termination

• Little is known 3 ends are removed immediately,
  which makes them difficult to study
• Very long trailer regions (1000 2000 bp)

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RNA Synthesis gt Transcription gt E.Coli
RNA polymerase I, III

• Differences in promoters
• RNA polymerase III, sometimes within gene