Eukaryotic RNA polymerases

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Biochemistry 441Lecture 10Ted YoungMarch 3,
2006

• Eukaryotic RNA polymerases
• Structure
• Function
• Complexity of polII transcription

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Three eukaryotic RNA polymerases
RNA

• 1. Pol I nucleolar, ribosomal RNA.
  Amanitin-resistant.
• 2. Pol II nucleoplasm mRNA precursors.
  Amanitin-sensitive.
• 3. Pol IIInucleoplasm small nuclear and
  cytoplasmic RNAs. amanitin-sensitive.
• (4. Mitochondrial)

DNA
Electron crystallography
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Some generalities about eukaryotic transcription

• 1. None of the purified RNA polymerases can
  initiate transcription.
• 2. Specific initiation requires additional
  specific transcription factors.
• 3. Activated transcription requires additional
  specific as well as general factors.

Why is eukaryotic transcription so much more
complicated than transcription in
bacteria? Possible answers 1. More DNA (but
yeast ). 2. Multi-cellular ( but yeast ). 3.
Complex regulation-integrating signals. 4.
Nuclear location. 5. Condensed in nucleosomes.
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Subunit structure of eukaryotic RNA polymerases
pol I pol II pol III
Note 1. Homology of the core subunits to
bacterial RNA polymerase subunits a, b, and
b. 2. Homologous subunits between polymerases
I, II, and III. 3. Common subunits. 4. Specific
subunits
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Pol I transcriptiongtribosomal RNA
Non-transcribed region

• Transcribed region

Non-transcribed spacer
)n
(
n/gt100
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Pol III transcriptiongttransfer RNA and other
small RNAs involved in RNA processing
Promoter
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DNA
RNA precursor
100-300 nucleotides
Final product tRNA
75-200 nucleotides
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Pol II transcriptiongtmessenger RNA
(protein-coding transcripts)
Enhancer elements
Core or basal promoter
Promoter
5 UTR (untranslated region 3 UTR
Ex In ExIn Ex
Precursor RNA
O R F
5
3
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Pol II transcription continued
Core promoter TATA box (analogous to -10 region
in E. coli promoters) initiator element (1
site).
Enhancers Distance and orientation independent
sequences that determine the efficiency and
specificity of the promoter.
TATA box
1
27 ntds
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Pol II transcription continued
Proteins required for transcription and possible
order of function
1. A sequence-specific DNA binding protein, a
transcription factor, binds the enhancer (not
shown). 2. This enhances binding of TATA- binding
protein (TBP) 3. Other general transcription
factors (GTFs) are recruited, either alone or
as part an RNA polymerase holoenzyme to form a
pre-initation complex (PIC). p11
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Pol II transcription continued some more
The pre-initiation complex (PIC) can be assembled
without an enhancer, or a transcription factor
(T1-T4), or without the mediator complex, but in
any of these situations initiation is slow and
inefficient.
4. A coactivator or Mediator contacts both the
activator and the PIC
Mediators (of the interaction between
activators and the PIC) are themselves
multi-subunit proteins
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Unusual sequence at carboxy-terminus of largest
subunit of pol II

• Pol II largest subunit from all organisms
  terminates with an unusual repeated heptapeptide
  (YSPTSPS)n CTD (Carboxy-terminal domain)

humans 52 repeats Drosophila 44
repeats Arabidopsis (plant) 41
repeats Caenohabditis (nematode) 34
repeats Saccharomyces (yeast) 26 repeats
Phosphorylation of the CTD regulates several
steps of transcription
CTD(bound) gt CTD-P(ser5)(initiated) gtCTD-P
(ser2)(elongating)
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Summary-transcription in eukaryotes

• Transcription of nuclear eukaryotic genes
  requires three related RNA polymerase
  enzymes-polI (pre-rRNA), polII (pre-mRNA,
  microRNA), and polIII(pre-tRNA, snRNA)
• Initiation of transcription requires general and
  specific transcription factors
• Promoter clearance and transcription elongation
  by polII is regulated by phosphorylation of its
  repetitive carboxy-terminal domain.