Eukaryotic RNA polymerases

1
(No Transcript)
2
Biochemistry 441Lecture 12Ted YoungFebruary 6,
2008

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

3
Imagination is more important than knowledge,
for knowledge is limited while imagination
embraces the entire world Albert Einstein
4
Three eukaryotic RNA polymerases
RNA

• 1. Pol I nucleolar, synthesizes 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
5
Some generalities about eukaryotic transcription

• 1. None of the purified RNA polymerases can
  initiate transcription.
• 2. Initiation requires additional proteins called
  transcription factors).
• 3. Activated transcription requires additional
  protein factors (there are 3000 encoded in the
  human genome, representing 10 of all genes
• 4. Overcoming repression by nucleosomes requires
  modification of histones as well as displacement
  of nucleosomes by ATP-dependent protein
  complexes.
• Transcription of a single gene by polII requires
  100 proteins!

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.
6
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 These facts imply that all
three polymerases arose from a common ancestral
polymerase.
7
Pol II transcriptiongtmessenger RNA
(protein-coding transcripts)
enhancers core promoter
RNA pol II and many transcription factors
8
The basal or core promoter of genes transcribed
by RNA pol II
Variable distance to non-TATA motifs
Constant distance to TATA motif 30 bp
Core promoter
gene
Binding sites or motifs Short, specific DNA
sequences different genes have different
combinations of motifs.
Each motif is bound by a transcription factor
Diagram based on and adapted from Figure 28.26 of
Mathews van Holde, Biochemistry, 2nd ed.
9
The TATA box and TATA box binding protein, Tbp.
TATA box
1
27 ntds
10
Structure of human Tbp
stirrup
DNA
saddle
11
Structure of human TbpBrookhaven Protein
Database ID1TGH
DNA is bent about 60o
12
Pol II transcription
Purified RNA polII is inactive on duplex
DNA. Initiation on duplex DNA requires protein
complexes called general transcription factors
(GTFs). GTFs allow RNA pol II to bind in the
absence of all four NTPs and form a
pre-initiation complex (PIC). In the presence of
all four NTPs initiation occurs at a promoter but
transcription is very weak.
PIC
13
Model of TFIID based on cryo-EM
14
Proposed structure of the pre-initiation complex
Benoit Coulombe's laboratory at the Clinical
Research Institute of Montreal
15
Activated transcription
DNA sequence-specific transcription factors (TFs
T1-T4 in diagram below), bind to the DNA and
recruit RNA polII, GTFs, and coactivators to
the promoter and increase the rate of initiation.
A coactivator or Mediator ( ) contacts
both the activator and the PIC. Mediator (of
the interaction between activators and pol II) is
itself a multi-subunit (15-20) protein complex
that is conserved in all eukaryotes. Mediator was
discovered in much the same way that E. coli
Sigma factor was discovered as a necessary
factor for promoter recognition by E. coli RNA
polymerase
16
Interaction between TF and polII by looping of
the DNA
Diagram based on and adapted from Fig. 1 of
Struhl, K., Cell 84 179-182
17
RNA polII carboxy-terminal domain (CTD)

• Pol II largest subunit 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 The CTD
is essential for transcription.
18
CTD phosphorylation leads to promoter clearance
G. Orphanides and D. Reinberg (2002) A Unified
Theory of Gene Expression. Cell 108 439-451.
19
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.
• Elongation of transcription requires additional
  protein complexes as well as movement of
  nucleosomes by chromatin remodeling complexes
• Promoter clearance and transcription elongation
  by polII is regulated by phosphorylation of its
  repetitive carboxy-terminal domain.