Ribonucleoprotein delivery of CRISPR-Cas9 reagents for increased gene editing...
FinalPresentation
1. Elongation Factor IV
Connor Stewart & Eric Newman
Laboratory of Biochemistry, Bellingham Washington
http://myhome.sunyocc.edu/~weiskirl/parts_of_all_cells.htm
2. Elongation Factor IV / Lep A
•High level of conservation
-Found in all sequenced prokaryotes and nearly all eukaryotes
•Name change from Lep A to Elongation factor 4 (EF4)
-Named Leading peptidase A due to it’s location on the Lep operon
-EF4 back-translocase function during elongation found using 32P labeling
•Stored on E. coli periplasmic membrane
- 1/5 Cytoplasm/Membrane
-Unique C terminal domain (CTD)
•G protein based
-Uncoupled ribosome dependent GTPase activity
-turnover rate similar to EF-G
PECH, MARKUS. KARIM, ZHALA. ET AL. (2010). P.N.A.S. VOL. 108(8), 3199-3203.
QIN, YAN. POLACEK, NORBERT. ET (2006). CELL VOL. 127(4), 721-733.
3. EF4 & EF-G Homology
QIN, YAN. POLACEK, NORBERT. ET (2006). CELL VOL. 127(4), 721-733.
• Strong homology between domains I, II, and III
and V of EF-G
• Domain configuration conserved from Yeast
through Humans
• EF-G, EF-Tu, IF2 all share homology with EF-4
• EF-4 retains 55-68% amino acid identity among
bacterial orthologs
• EF-G retains 58-70% amino acid identity
5. GAGNON G., MATTHIEU. LIN, JINZHONG. ET AL. (2014) SCIENCE, VOL.345(6197), 684-687.
EF-4 Bound to Ribosome
• Competitive binding with EFG
• CTD inserts into A-site and
connects to post translational
complex
-last 44 residues not visible
6. Function
•Released during unfavorable conditions
-High ionic strength, low temperatures. Addition of Mg2+ changes ratio from 5/1 to 1/5
membrane/cytoplasm
-Addition of [2-5] Mg2+ reduces GFP synthesis by 40%
-Active GFP from 50% - 25%
-EF4 addition brings GFP synthesis to 120%
-Active GFP maintained at 50%
•Catalyzes the back-translocation reaction on post-translocation state ribosomes.
-Reverses EF-G catalyzed transition, giving EF-G a second chance at correct t-RNA translocation
-Re-mobilizes stuck ribosomes
GAGNON G., MATTHIEU. LIN, JINZHONG. (2014) SCIENCE, VOL.345(6197), 684-687.
PECH, MARKUS. KARIM, ZHALA. (2010). P.N.A.S. VOL. 108(8), 3199-3203.
7. Mechanism
•Competes with Elongation Factor G
-EF4 has no EFG domain IV backstop and reduces post – pre conformation energy
barrier.
Preferentially binds to Post-Ribosomal Complex
-Pb2+ cleavage of engineered pre and post ribosomal complexes
-17.8% of PRE complexes cleaved, 82.2% of POST cleaved
-Addition of EF4 brought POST cleavage back to PRE levels
-shifts tRNA from E&P sites to P&A
•Back ratcheting re-opens A-site giving EFG a second chance
- 32P labeling of Amino Acids, one codon length back
GAGNON G., MATTHIEU. LIN, JINZHONG. ET AL. (2014) SCIENCE, VOL.345(6197), 684-687.
9. Cell Optical Density during Incubation
0 25 50 75 100
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
OpticalDensity
Time (min)
Optical Density (A.U.) of incubating cells as a
function of time (minutes).
• Incubated at 15oC overnight with IPTG for
overexpression
10. Chromatography Elution Curve
0 2 4 6 8 10 12 14 16 18 20
0
5
10
15
20
25
30
Absorbance(A.U.)
Protein Fraction
• Absorbance at 280 nm of affinity
chromatography elution fractions.
• Fractions 2-4 were diluted 1/100 for absorbance
readings.
• The values displayed for fractions 2-4 account
for this dilution.
11. Dialysis
EF-4 precipitated during dialysis
Procedure Modification:
• Centrifuged on Eppendorf minispin® at 12,000 x g
• Absorbance was measured at 280 nm and supernatant was used for future analysis.
12. Bradford Assay
0 2 4 6 8 10 12 14 16 18 20 22
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Absorbance(A.U.)
BSA (g)
Linear Fit Parameters:
m = 0.03701
b = 0.03395
R2
= 0.98832
• Bradford Assay standard curve. Absorbance
(A.U.) taken at 595 nm plotted as a function of
bovine serum albumin (BSA) µg with a linear
fit.
• A 40 µg point was excluded to maintain the
linear fit.
• Mass extinction coefficient of 3.56 L/(g∙cm).
• Literature Value: 0.593 L/(g∙cm)
• Protein concentration 0.57µg/µL
16. Determination of Unknown E
•Gel electrophoresis: 69 kDa
•1.6 kDa off of the Literature value (2.4%)
• Walter D., Justin. Littlefield, Peter. Delbecq, Scott. Prody, Gerry. Spiegel P, Clint. (2010). Expression, purification, and
analysis of unknown translation factors from Escheria coli: A synthesis approach. Biochemistry and Molecular
Biology Education. Volume 38(1), 17-22
21. CD-Spectra
40 45 50 55 60
-30
-28
-26
-24
-22
-20
-18
Temperature (o
C)
R2
= 0.969
y = 1.19x - 80.9
R2
= 0.543
y = -0.134x - 12.4
R2
= -0.656
y = 0.0630x - 31.6
Ellipticity(mdeg)
22. CD Thermodynamics
0.00308 0.00310 0.00312 0.00314 0.00316
-4
-2
0
2
4
6
ln(Keq
)
1/T (K-1
)
R2
= 0.971
y = -103904x + 323.98
• Possible source of error: low protein
concentration
ΔH 860 kJ/mol
ΔS 2.7 kJ/mol
ΔGo’ 61 kJ/mol
23. Further Studies
Aggregation
• We feel the precipitation of our protein may have skewed some of our results and experiments.
• Experiments to determine more stable conditions for EF4
• More accurate information would most likely be achieved by performing experiments in a
higher ionic environment and/or a lower temperature.
Mechanism to explore
• Binding to periplasmic membrane
• Types of interactions with and conformation changes to the ribosome.
