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BackgroundBackground• Biology and Biomedicine are rapidly evolving
from 'data-poor' to 'data-rich'-sciences
Genomics
Transcriptomics
ProteomicsMetabolomics
Interactomics
Glycomics
Lipidomics
BackgroundBackground
• The new technologies share a common property:
• High-throughput technologies are essential for describing and cataloguing the complexities of biological systems
• Basic Science: important for functional insights
• Diagnostics: allows detection of normal and abnormal states
High-Throughput
www.sys-bio.org/contentimages/
BioinformaticsBioinformatics
• The large data sets reveal functional correlations between individual systems elements
• Enhanced understanding of complex systems
• Computer simulations can be used to test predictions against real data sets
New ChallengesNew Challenges
• ‘Systems approaches’ have been successfully applied to a number of biological systems
• There are, however, major gaps at the most fundamental level: Molecular structure/function relationships are still poorly understood!
Molecular Molecular Structure/Function Structure/Function
RelationshipsRelationships
Catalytic Center of
RNA Polymerase II
Tan, L., Wiesler, S., Trzaska, D., Carney, H.C. and Weinzierl, R.O.J. (2008). Bridge helix and trigger loop perturbations generate superactive RNA polymerases. J. Biol. 7, 40.
Structure/Function Studies Structure/Function Studies
• Structural Approaches:• X-ray crystallographic analysis of bacterial,
archaeal and yeast RNAPs
• Genetic Approaches:• Isolation of random mutants, especially in bacterial and
yeast RNAPs, displaying detectable phenotypes
• Biochemical Approaches:• Chemical cross-linking studies; nucleotide analogs
Roger KornbergNobel Prize in Chemistry, 2006
• single snapshots; ‘crippled’ complexes containing non-functional substrates;
• only certain mutants display detectable phenotypes; stability/ viability issues
• time-scale and/or specificity often difficult to controlLo
w Thr
ough
put!
Mutagenesis with
one oligonucleotide
Amplification and transfer into expression host
High-throughput plasmid purification & sequencing
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Saturation Mutagenesis - ExamplesSaturation Mutagenesis - Examples
mjA' A822-X
mjA' Q823-X
T821
ACC
A822
GCG
Q823
CAG
R820
CGT
S824
AGC
G825
GGT
Y826
TAT
V819
GTG
A818
GCG
• The likelihood of obtaining particular substitutions depends on the frequency of codons within the genetic code
Werner, F., and Weinzierl, R.O.J. (2002). A recombinant RNA polymerase II-like enzyme capable of promoter-specific transcription. Mol. Cell 10, 635-646.Ouhammouch, M., et al. (2004). A fully recombinant system for activator-dependent archaeal transcription. J. Biol. Chem. 279, 51719-51721.Werner, F., and Weinzierl, R.O.J. (2005). Direct modulation of RNA polymerase core functions by basal transcription factors. Mol. Cell. Biol. 25, 8344-8355.
Archaeal RNAP(Methanocaldococcus jannaschii)
Mix in 6M urea and dialyze to assemble native RNAP
The RNA Polymerase FactoryThe RNA Polymerase Factory
IN:1.5 ml bacterial cultures expressing
different mutant subunits
OUT:Purified and characterized mutant recombinant subunits
OUT:Recombinant RNAPs assembled with the mutant subunits purified from bacterial cultures
OUT:Expression plasmids archived for long-term storage
OUT:High-throughput activity measurements from in vitro transcription results
Nottebaum, S., Tan, L., Trzaska, D., Carney, H.C., and Weinzierl, R.O.J. (2008). The RNA polymerase factory: a robotic in vitro assembly platform for high-throughput production of recombinant protein complexes. Nucl. Acids Res. 36, 245-252.
Protein quantitationBCA assay
Cell culturesAutoinduction medium
Protein extractionFastBreak/Lysonase
ChromatographyIon exchange and affinity
96-well Assembly of RNAP
96-well Transcription assays
HT electrophoresisE-PAGE48/E-PAGE96
DNA/RNA quantitation
Fluorescent assaysSubunit archiving
Barcoded storage (-80oC)
Cell density quantitationA600 assay
Viability quantitationPropidium iodide assay
Clone archivingWhatman FTA cards
RNAP archivingBarcoded storage (-80oC)
Dialysis efficiencyFluorescent assay
Specific trx assaysBarcoded storage (-80oC)
Non-specific trx assaysFluorescent assay
STAGE
2
STAGE
3
STAGE
1
E-PAGE 96
Bacterial Cell Density
Subunit Concentration
% Non-viable Cells
Expression Strain Identity
Individual
2D Barcode
Parallel Robotic Assembly of 96 Parallel Robotic Assembly of 96 Different RNAPsDifferent RNAPs
DialysisMembrane
Urea-freeBuffer
Waste
Magnetic Stirrer
Hours
[Urea](Molar)
wt
mjRNAP (wildtype)
V819A
mjRNAP A'-V819A
R820AmjRNAP A'-R820A
etc. (x96!)
T821A
mjRNAP A'-T821A
Fu
nction
al Assays
• Complete mutagenesis data for 17 successive amino acid positions reveals a wide variety of phenotypes
• Identification of the most informative mutants for revealing reaction mechanism
• Approach also identifies mutants that do not have a significant effect
T821
A822
Q823
A822 Side Chain Requirements
• (variable; large hydrophilic side chains [Q, R] acceptable)
Q823 Side Chain Requirements
• (quite variable)
• direct control of catalytic rate
ConclusionsConclusions
• Robotic applications in Molecular Biology do not make life easier – they expand what can be done
• Complex experiments, once automated, can produce more results than humanly (psychologically!) possible
• Repeats and multiple samples provide statistical measure of accuracy/reproducibility
• The collection of large systematic data sets allow the unbiased detection of unexpected phenomena
mjRNAP
neRNAP
Fluoride salts
wt activity
High-throughput Transcription AssaysHigh-throughput Transcription Assays