Transcript Poised RNA polymerase

Poised RNA polymerase
Debate
Positive: Microarray analysis for determining the level
of accumulation of mRNAs is an efficient
methodology for identifying proteins that contribute to
the process being studied.
Negative: Not
Debate Format
Pro
Con
3 minutes state case for affirmative
1 min consult 2 min rebuttal
1 min consult 2 min rebuttal
3 minutes state case against
1 min consult 2 min rebuttal
1 min consult 2 min rebuttal
1 min consult 2 min rebuttal
1 min consult 2 min rebuttal
2 min Conclusion for Pro
2 min Conclusion for Con
1 min consult 2 min rebuttal
1 min consult 2 min rebuttal
Lactose repressor
LacI
RNAP
8 base abortive transcript
Chromatin Immunoprecipitation
UV irradiate
Chromatin Immunoprecipitation
Protein crosslinking to DNA
Chromatin Immunoprecipitation
Extract Protein and DNA complexes
Chromatin Immunoprecipitation
Fragment DNA
Chromatin Immunoprecipitation
Immunoprecipitate with antibody to RNAP
or your protein of choice.
Chromatin Immunoprecipitation
Lis paper
Detect DNA with a labelled probe
Three potential results
Poised RNA polymerase
Heat shock promoter uninduced
HSF
RNAPII
20-30 base transcript
HSF
RNAPII
20-30 base transcript
LacI
RNAP
8 base abortive transcript
Instead of looking at one gene or one car
Look at the genome
or highway.
Chromatin Immunoprecipitation
on chip
Label DNA and hybridize to a chip with a
tiled array of genomic sequences
Three potential results
Poised RNA polymerase
E. coli Genome
23% of genes have poised RNA polymerases
Drosophila S2 cells
7,702
3,089
1,014
Drosophila embryos
37%
27%
12%
Drosophila embryos
The 12% of genes that have a poised RNAPII are
enriched for developmental genes
Heat shock promoter uninduced
HSF
RNAPII
20-30 base transcript
Sequencing 8.4 million short RNAs
Poised promoter
20-30 base transcript
RNAPII
RNAPII
20-30 base transcript
Drosophila embryos
37%
27%
12%
Drosophila embryos
The 12% of genes that have a poised RNAPII are
enriched for developmental genes
Drosophila embryos
Snail is a repressor bound at 139 genes
These genes are repressed in Toll10b mutants
54% have poised RNAP II (P<10-23)
The three ‘habits’
• Barolo and Posakony 2002
• Looking for common themes in the
organization of signaling pathways.
Trying to explain the precision of expression
Example wingless expression
The three ‘habits’
• Activator insufficiency
• Cooperative activation
• Default repression
SPRE: Signaling pathway response elements
SPRE-binding
transcription factor
Other factors
Barolo and Posakony Genes and Dev. 16, 1167
Basis for the proposal of the model
Barolo and
Posakony Genes
and Dev. 16, 1167
Activator insufficiency
• SPRE-binding transcription factor can
not activate transcription alone.
• Tissue culture vs in vivo
Cooperative activation
• SPRE-binding transcription factors
require other transcription factors for the
activation of transcription.
• The interaction is cooperative
Default repression
• In the absence of ligand SPRE-binding
factors repress transcription.
3 habits model
Barolo and Posakony Genes and Dev. 16, 1167
Consequences of the 3 ‘habits’
Barolo and Posakony Genes and Dev. 16, 1167
Default repression
• In the absence of ligand SPRE-binding
factors repress transcription.
• Is the default repression mechanism
stalling the RNA polymerase?