Transcript The Archaeal Transcriptional Machinery

Automation of Complex Procedures in
Molecular Biology
Robert Weinzierl
Imperial College London
Background
• Biology and Biomedicine are rapidly evolving
from 'data-poor' to 'data-rich'-sciences
Lipidomics
Genomics
Genomics
Transcriptomics
Interactomics
Metabolomics Glycomics
Proteomics
Background
• The new technologies share a common property:
High-Throughput
• 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
Bioinformatics
• 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 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
Structure/Function
Relationships
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
• Structural Approaches:
• X-ray crystallographic analysis of bacterial,
archaeal and yeast RNAPs
• single snapshots; ‘crippled’ complexes containing nonfunctional substrates;
• Genetic Approaches:
Roger Kornberg
Nobel Prize in Chemistry, 2006
• Isolation of random mutants, especially in bacterial and
yeast RNAPs, displaying detectable phenotypes
• only certain mutants display detectable phenotypes; stability/ viability issues
• Biochemical Approaches:
• Chemical cross-linking studies; nucleotide analogs
• time-scale and/or specificity often difficult to control
1. Select your Mutagenesis Target …
The Bridge Helix
PDB #1R5U and 1I6H
2. Prepare lots of mutants …
http://www.suta-raito.com/Neopets.shtml
Mutagenesis with
one oligonucleotide
Amplification and transfer
into expression host
High-throughput plasmid purification & sequencing
HT Targeted Mutagenesis
Sequencing and
RNAP Factory
X
NNN
NNN
Saturation Mutagenesis - Examples
mjA' A822-X
mjA' Q823-X
• The likelihood of obtaining
particular substitutions depends
on the frequency of codons
within the genetic code
A V R T A Q S G Y
818 819 820 821 822 823 824 825 826
GCGGTGCGT ACCGCG CAGAGCGGT TAT
3. Prepare mutant subunits
and incorporate them into an intact
enzyme …
Archaeal RNAP
(Methanocaldococcus jannaschii)
Mix in 6M urea and dialyze
to assemble native RNAP
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.
The RNA Polymerase Factory
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:
IN:
1.5 ml bacterial cultures expressing
different mutant subunits
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.
Cell cultures
Autoinduction medium
Cell density quantitation
A600 assay
Clone archiving
Whatman FTA cards
Viability quantitation
Propidium iodide assay
STAGE
1
Protein extraction
FastBreak/Lysonase
Protein quantitation
BCA assay
DNA/RNA quantitation
Fluorescent assays
HT electrophoresis
E-PAGE48/E-PAGE96
Subunit archiving
Barcoded storage (-80oC)
96-well
Assembly of RNAP
Dialysis efficiency
Fluorescent assay
RNAP archiving
Barcoded storage (-80oC)
STAGE
2
96-well
Transcription assays
Non-specific trx assays
Fluorescent assay
Specific trx assays
Barcoded storage (-80oC)
STAGE
3
Chromatography
Ion exchange and affinity
Expression Strain Identity
E-PAGE 96
Individual
2D Barcode
Bacterial Cell Density
6
A600
5
4
3
2
1
90
% non-viable
80
70
60
50
40
30
20
10
0
micrograms/microliter
1.2
1
0.8
0.6
0.4
0.2
0
Subunit Concentration
H
7
7
H
1
1
% Non-viable Cells
G
1
7
F
F
E
7
E
1
D
7
D
1
C
7
C
1
B
7
B
1
G
100
A
7
A
1
0
Parallel Robotic Assembly of 96
Different RNAPs
6.000
5.000
4.000
[Urea]
(Molar)
Series
3.000
Series
2.000
1.000
0.000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Hours
Dialysis
Membrane
Urea-free
Buffer
Waste
Magnetic Stirrer
mjRNAP (wildtype)
wt
V819A
mjRNAP A'-R820A
R820A
mjRNAP A'-T821A
T821A
etc. (x96!)
Functional Assays
mjRNAP A'-V819A
• 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
Q823
Q823 Side Chain Requirements
• (quite variable)
• direct control of catalytic rate
A822
T821
A822 Side Chain Requirements
• (variable; large hydrophilic side
chains [Q, R] acceptable)
Conclusions
• 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
Further Details
BBSRC
High-throughput Transcription Assays
mjRNAP
neRNAP
Fluoride
salts
wt activity
neRNAP is a 'Fluorophile '
wt activity
[10x]
Tris-Ac
Tris-HCl
Tris-Ac
+PEG
Tris-HCl
Potassium fluoride
Ammonium fluoride
Potassium fluoride
Ammonium fluoride
+PEG
+ 0.1M Tris-HCl pH8.5
+ 0.1M Tris-HCl pH8.5
+25% PEG3350
+25% PEG3350