Elongation Factor IV

Connor Stewart & Eric Newman

Laboratory of Biochemistry, Bellingham Washington

http://myhome.sunyocc.edu/~weiskirl/parts_of_all_cells.htm

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-GPECH, 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.

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

Visualized in PyMOL

90o

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

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.

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.

Transformation Efficiency Volume Colonies Transformation efficiency

100 µL Native E. coli 0 0 colonies/µg

50 µL 0 0 colonies/µg

100 µL 2 1000 colonies/µg

200 µL 0 0 colonies/µg

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

Opt

ical

Den

sity

Time (min)

Optical Density (A.U.) of incubating cells as a function of time (minutes).

• Incubated at 15oC overnight with IPTG for overexpression

Chromatography Elution Curve

0 2 4 6 8 10 12 14 16 18 20

0

5

10

15

20

25

30

Abs

orba

nce

(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.

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.

Bradford Assay

0 2 4 6 8 10 12 14 16 18 20 220.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Abs

orba

nce

(A.U

.)

BSA (g)

Linear Fit Parameters: m = 0.03701b = 0.03395R2 = 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

#1 #2 #3 #4 #5 #6 #7 #8 #9 #10

SDS-PAGE

• #1 Cell Lysate Supernatant• #2 Crude Cell Lysate• #3 Purified Lysate with Aggregate • #4 Chromatography High-speed flow through• #5 Chromatography Fraction 1• #6 Purified Cell Lysate• #7 Molecular Weight Ladder• #8 Chromatography Rinse 2• #9 Chromatography Rinse 1• #10 Dialysis Buffer

0.0 0.2 0.4 0.6 0.8 1.0

20

40

60

80

100

Mol

ecul

ar W

eigh

ts (

kDa)

Relative Migration

Log(y) = 2.23 + (-2.77)x1 + (3.56)x2 + (-2.2)x3

R2 = 0.994

Relative Mobility

• Experimental M.W.: 69 kDa

• EF4 molecular weight: 67.393 kDa

Relative Migration of protein standards against log10 molecular weights.

Determination of Unknown ETranslation Factor

Molecular Weight (kDa)

Isoelectric Point (pI)

Mass extinction coefficient (L∙g-1∙cm-

1)

IF2 98.2 4.97 0.280

IF3 21.3 9.54 0.209

EF-Tu 44.1 5.6 0.465

EF-G 78.4 5.43 0.784

EF-4 67.4 5.68 0.593

RRF 21.4 7.03 0.139

RF1 41.3 5.40 0.521

RF3 60.4 5.91 0.691

• Mass extinction coefficient of 3.56 L/G∙cm.

• M.W.: 69 kDa

• Aggregation

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

Fluorimetry

300 325 350 375 4000

500

1000

1500

2000

2500

Inte

nsity

(A

.U)

Wavelength

Fluorimetry

0 1 2 3 4 5 61500

1600

1700

1800

1900

2000

2100

2200

2300

2400

R2 = 0.99458.7x + 2200

R2 = 0.953y = -182x + 2688

R2 = -0.755y = 22.0x + 1600

Inte

nsity

(A

.U.)

Urea (M)

Fluorimetry Thermodynamics

• ΔGo' = 16 kJ/mol

•Tertiary structure

2.5 3.0 3.5 4.0 4.5 5.0

-10000

-8000

-6000

-4000

-2000

0

2000

4000

6000

Gib

bs F

ree

Ene

rgy

(J/m

ol)

Urea (M)

R2 = 0.824y = -4663x + 16280

CD-Spectra

215 220 225 230 235 240-35

-30

-25

-20

-15

-10

-5

0

Elli

ptic

ity (

mde

g)

Wavelength

CD-Spectra

40 45 50 55 60-30

-28

-26

-24

-22

-20

-18

Temperature (oC)

R2 = 0.969y = 1.19x - 80.9

R2 = 0.543y = -0.134x - 12.4

R2 = -0.656y = 0.0630x - 31.6

Elli

ptic

ity (

mde

g)

CD Thermodynamics

0.00308 0.00310 0.00312 0.00314 0.00316

-4

-2

0

2

4

6

ln(K

eq)

1/T (K-1)

R2 = 0.971y = -103904x + 323.98

• Possible source of error: low protein concentration

ΔH 860 kJ/mol

ΔS 2.7 kJ/mol

ΔGo’ 61 kJ/mol

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.

Acknowledgements

•Gerry Prody

•Clint Spiegel

•Michelle Wuerth

•Marcus Carlson

Thank You

Questions?

QIN, YAN. POLACEK, NORBERT. ET AL. (2006). CELL VOL. 127(4), 721-733.