Protein
Protein
Protein
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Towards Quantitative Biology<br />
Robin Friedman
ATG ACG AAG...<br />
Methionine<br />
Lysine<br />
Threonine<br />
DNA<br />
mRNA<br />
<strong>Protein</strong>
ATG ACG AAG...<br />
Methionine<br />
Lysine<br />
Threonine<br />
DNA<br />
DNA<br />
DNA<br />
mRNA<br />
mRNA<br />
mRNA<br />
<strong>Protein</strong><br />
<strong>Protein</strong><br />
<strong>Protein</strong>
ATG ACG AAG...<br />
Methionine<br />
Lysine<br />
Threonine<br />
DNA<br />
DNA<br />
DNA<br />
mRNA<br />
mRNA<br />
mRNA<br />
<strong>Protein</strong><br />
<strong>Protein</strong><br />
<strong>Protein</strong><br />
One gene One protein One function
Number of genes<br />
Organismal complexity
Species<br />
Number of Neurons Number of genes<br />
Human 10 11 21,000<br />
Mouse 10 7 22,000<br />
Fruitfly 10 5 14,000<br />
Nematode 302 19,000
Species<br />
Number of Neurons Number of genes<br />
Human 10 11 21,000<br />
Mouse 10 7 22,000<br />
Fruitfly 10 5 14,000<br />
Nematode 302 19,000
Species<br />
Number of Neurons Number of genes<br />
Human 10 11 21,000<br />
Mouse 10 7 22,000<br />
Fruitfly 10 5 14,000<br />
Nematode 302 19,000<br />
1.5% of human genome is protein coding
Nucleus<br />
Cytoplasm<br />
DNA<br />
pre-mRNA<br />
transcription<br />
<strong>Protein</strong><br />
splicing<br />
mRNA<br />
translation<br />
export<br />
degradation
Nucleus<br />
Cytoplasm<br />
DNA<br />
pre-mRNA<br />
transcription<br />
<strong>Protein</strong><br />
mRNA<br />
splicing<br />
translation<br />
miRNAs<br />
export<br />
degradation
Transcription<br />
Factors<br />
RNA Binding<br />
<strong>Protein</strong>s<br />
mRNAs<br />
miRNAs
Transcription<br />
Factors<br />
RNA Binding<br />
<strong>Protein</strong>s<br />
mRNAs<br />
miRNAs
Transcription<br />
Factors<br />
RNA Binding<br />
<strong>Protein</strong>s<br />
mRNAs<br />
miRNAs
Sequencing cost (nucleotides/dollar)<br />
10 6<br />
10 4<br />
10 2<br />
10 0<br />
10 6<br />
10 4<br />
10 2<br />
10 0<br />
Storage cost (megabytes/dollars)<br />
1990 1995 2000 2005 2010<br />
Year
Transcription<br />
Factors<br />
RNA Binding<br />
<strong>Protein</strong>s<br />
mRNAs<br />
miRNAs
Electrophoretic mobility shift assay<br />
Bound DNA<br />
Unbound DNA<br />
Increasing protein concentration
Electrophoretic mobility shift assay<br />
Bound DNA<br />
Unbound DNA<br />
Increasing protein concentration
10 Gb per run<br />
Illumina Genome Analyzer
Illumina Genome Analyzer<br />
GCC AGA TTG<br />
>10 Gb per run
Illumina Genome Analyzer<br />
GCC AGA TTG<br />
A<br />
C<br />
G<br />
T<br />
>10 Gb per run
Illumina Genome Analyzer<br />
GCC AGA TTG<br />
A<br />
C<br />
G<br />
T<br />
>10 Gb per run
Illumina Genome Analyzer<br />
GCC AGA TTG<br />
A<br />
C<br />
G<br />
T<br />
>10 Gb per run
Illumina Genome Analyzer<br />
GCC AGA TTG<br />
A<br />
C<br />
G<br />
T<br />
>10 Gb per run
Illumina Genome Analyzer<br />
A channel
Illumina Genome Analyzer<br />
A channel<br />
C channel<br />
G channel<br />
T channel
Illumina Genome Analyzer<br />
A channel<br />
C channel<br />
Merge<br />
G channel<br />
T channel
HiTS-FLIP method<br />
High-Throughput Sequencing<br />
Fluorescent Ligand Interaction Profile<br />
100,000,000<br />
Random 25mers
HiTS-FLIP method<br />
High-Throughput Sequencing<br />
Fluorescent Ligand Interaction Profile<br />
100,000,000<br />
Random 25mers
HiTS-FLIP method<br />
High-Throughput Sequencing<br />
Fluorescent Ligand Interaction Profile<br />
100,000,000<br />
Random 25mers<br />
GCN4<br />
mOrange tag
HiTS-FLIP method<br />
High-Throughput Sequencing<br />
Fluorescent Ligand Interaction Profile<br />
100,000,000<br />
Random 25mers<br />
GCN4<br />
mOrange tag
GCN4<br />
HiTS-FLIP method
HiTS-FLIP method<br />
GCN4<br />
Merge
HiTS-FLIP method<br />
GCN4<br />
Merge
HiTS-FLIP method<br />
GCN4<br />
Merge
GCN4<br />
master regulator of amino<br />
acid biosynthesis in yeast
GCN4<br />
master regulator of amino<br />
acid biosynthesis in yeast<br />
Decades of research<br />
on binding specificity<br />
ATGACTCAT<br />
ATGAGTCAT
GCN4 binds its canonical motif<br />
440,000,000 data points
GCN4 binds its canonical motif<br />
Intensity above background<br />
0.9<br />
0.8<br />
0.7<br />
0.6<br />
0.5<br />
0.4<br />
0.3<br />
0.2<br />
0.1<br />
0<br />
440,000,000 data points<br />
7mers<br />
Consensus:<br />
TATGACTCAT<br />
NNTGACTCAN<br />
NATGACTCNN<br />
NNTGACTAAN<br />
NGTGACTCNN<br />
TATGACTNNN<br />
NATGACTANN<br />
NATGACTGNN<br />
NNNGACTCAT<br />
GATGACTNNN<br />
NATGACACNN<br />
NNNTACAGGT<br />
NNNAGCTACC<br />
NGATACTANN<br />
NNNTCCTAAC<br />
NATGAATGNN
Concentration-dependent GCN4 binding<br />
Normalized wash intensity above background<br />
0.9<br />
0.8<br />
0.7<br />
0.6<br />
0.5<br />
0.4<br />
0.3<br />
0.2<br />
0.1<br />
0<br />
−0.1<br />
1 10 100 1000<br />
[Gcn4p] (nM)<br />
Consensus:<br />
TATGACTCAT<br />
NNTGACTCAN<br />
NATGACTCNN<br />
NNTGACTAAN<br />
NGTGACTCNN<br />
TATGACTNNN<br />
NATGACTANN<br />
NATGACTGNN<br />
NNNGACTCAT<br />
GATGACTNNN<br />
NATGACACNN<br />
NNNTACAGGT<br />
NNNAGCTACC<br />
NGATACTANN<br />
NNNTCCTAAC<br />
NATGAATGNN
Dissociation constants<br />
Normalized wash intensity above background<br />
0.9<br />
0.8<br />
0.7<br />
0.6<br />
0.5<br />
0.4<br />
0.3<br />
0.2<br />
0.1<br />
0<br />
10 0 10 1 10 2 10 3<br />
[GCN4] (nM)<br />
Hill coefficient = 2.0<br />
KD = 42 nM
Dissociation constants<br />
1000<br />
HiTS−FLIP K D<br />
(nM)<br />
100<br />
10<br />
10 100 1000<br />
Gel shift K D<br />
(nM)<br />
R. Nutiu
Binding interdependencies<br />
TGACTCA<br />
TGAGTCT
Conclusion
Conclusion<br />
Colected a half billion data points, recapitulated<br />
decades of GCN4 research in a single experiment
Conclusion<br />
Colected a half billion data points, recapitulated<br />
decades of GCN4 research in a single experiment<br />
Opens door for more quantitative modeling of<br />
transcription
Acknowledgements<br />
Razvan Nutiu, Christopher Burge,<br />
Gary Schroth, Shujun Luo
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