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Grabbing the Cat by the Tail: Studies of DNA Packaging by Single f 29 Bacteriophage Particles Using Optical Tweezers Acknowledgements Shelley Grimes Dwight Anderson University of Minnesota Sander Tans Douglas Smith Steven B. Smith Yann Chemla Aathi Karunakaran

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slide1
Grabbing the Cat by the Tail: Studies of DNA Packaging by Single f29 Bacteriophage Particles Using Optical Tweezers
acknowledgements
Acknowledgements

Shelley Grimes

Dwight Anderson

University of Minnesota

Sander Tans

Douglas Smith

Steven B. Smith

Yann Chemla

Aathi Karunakaran

University of California, Berkeley

bacteriophages
Bacteriophages

Icosahedral bacteriophageshave played an central

role in the development of Molecular Biology

Simplest infectious organisms known

Capsid: an empty protein shell that

contains the genetic material

of the phage

Tailand associated protein filaments

bacteriophage f 29
Bacteriophage f29

Volume of the capsid:

V ~ 56 x 10-3mm3

Length of f29 DNA:

19, 285 bp ~ 6.5 mm

dna confinement
DNA confinement

DNA is compacted about 6000x

inside the phage head

DNA concentration:~ 500 mg/ml

Opposing packaging:

electrostatic repulsion,

bending rigidity,

entropy loss,

dehydration.

DNA must be kept inside the bacteriophage head at

significant pressures.

the packaging motor
The packaging motor

• The head-tail connector (gp 10)

Mw = 36 KDa

Stoichiometry = dodecamer

Structure recently solved

other views of the connector
Other views of the connector

Top view with DNA model in channel

CryoEM reconstruction of Capsid

with connector crystal structure fit in

Side view: showing two monomers and DNA

Ref. Guasch A et al, JMB (2002)

the packaging motor cont d
The packaging motor (Cont’d)

• The packaging RNA (pRNA)

174 bases (57KDa) ,

Stoichiometry = 6mer (5mer)

(structure unknown)

the packaging motor cont d10
The packaging motor (Cont’d)

• An ATPase (gp 16)

Mw = 39 Kda

Stoichiometry = most likely 6/phage

Structure unknown

the atpase gp16
The ATPase (gp16)

gp16 – DNA dependent ATPase

objectives
Objectives

Characterize theForce vs. Velocityrelation of a

novel motor that may couple rotation to translation.

Determine thestall forceof the motor

Does aninternal pressurebuild up in the head? If

so, how much?

Where in the motor cycle does DNA translocation

occur?

What is the step size of the motor

slide15

Constant force

feed-back

No feed-back

Experimental Setup

packaging at constant force
Packaging at Constant Force

Video by Yann Chemla and Aathi Karunakaran

constant force 5 pn experiments
Constant force (5 pN) experiments

• Initial packaging rates~ 100 bp/sec.

• Pausesare frequent. Ave. pause duration:4 s ± 5 s.Neither

the pause duration nor the intervals between pauses are Poisson

distributed. Occur more often at higher fillings.

motor fluctuations
Motor fluctuations

Motor

Fluctuations

Noise

Observed rate variations are 5x larger than noise which is

~ 4 bp/s at 1 Hz bandwidth.

internal pressure
Internal Pressure

8 complexes

averaged &

smoothed

A single

complex

External force = 5 pN

• Rate decreases to zero as head fills up

• Up to 105 % of the f29 genome is packaged before stalling

• An internal pressuremust be building up due to DNA

confinement.

packaging without force feedback
Packaging without Force Feedback

Video by Yann Chemla and Aathi Karunakaran

slide23

Pipette & trap positions fixed

Trap & pipette positions fixed ->> LengthForce

Motor stalls at high force.

a powerful motor
A powerful motor

Average stall

force =55 pN

Max. force

meas.> 70 pN

force velocity relationship
Force-velocity relationship

Single complex traces:

- Stall force and initial speed vary

- Curve shapes are similar

Mean traces for 2 fillings:

F vs. velocity curves:

at1/3 filling

at2/3 fillingthe curve is

displaced to the left by~ 14pN

External

Force = 5 pN

1/3 filling

2/3 filling

force additivity
Force additivity

The good overlapobserved by shifting one curve

relative to the other suggests that theinternal

and external forcesacting on the motoradd.

Ext. and Int. forces

must be actingat the

same pointon motor.

slide27

Internal Force

No internal force in first half

Internal force ~50pN at completion

Pressure~6 MPa or 60 kg/cm2

slide29

Work done by the motor

Work done to package all DNA: 7.5x10-17 J

(2x104 kT or 8.2 x 104 pN nm)

Available energy per ATP : 120 pN nm

Maximum work done per ATP : 37 pN nm

(load = 55 pN; suppose step size=2 bp)

efficiency ~ 30%

(lower bound)

partitioning the work
Partitioning the work

Total work done by the motor = 8.2 x 104 pN nm

(or ~ 20,000 kBTs)

Ebending= EIq/2L = kBTP q/2L =2,180 pN nm (~ 530 kBTs)

Econfig. loss =900 pN nm (~ 220 kBTs)

Therefore, the dominant factor in the work

done by the motor appears to be theDNA

electrostatic self-repulsion and dehydration

the motor obeys michaelis menten kinetics
The motor obeys Michaelis-Menten kinetics

[T]n

(KM)n+[T]n

Hill coefficient n=1

V=Vmax

1 ATP hydrolyzed/cycle, no cooperativity between ATPases

f v relationships for various atp
F-v relationships for various [ATP]

V decreases monotonically vs. F, ATP

two regimes F<40pN, F>40pN

5mM ADP, 5mM Pi

less force dependence at low ATP

slide34

ATP

ATP

M1

M2

ADP

Pi

M3

ADP

Pi

Where is the translocation step?

k1

k2

k3

M1 +T <--> M2T --> M3D --> M1 + D

k-1

kcat =k2 k3/ (k2 + k3)~ Vmax

KM=(k2 +k-1) k3 / k1(k2 + k3)

Vmax/KM =(k2 +k-1)/(k1 + k2)

At low ATP, v = Vmax[T]/KM, binding is rate limiting: v depends on k1, k-1,k2 , independent of k3

At high ATP, v = Vmax, binding very fast: v depends on k2, k3, independent of k±1

Binding movement step:

k1 and k-1 are F dependent

Vmax force independent

Vmax/KM force dependent

KM force dependent

2. Reaction movement step:

k2 is F dependent

Vmax force dependent

Vmax/KM force dependent

KM force dependent

3. Release is the movement step:

k3 is force dependent

Vmax force dependent

Vmax/KM force independent

KM force dependent

binding

reaction

release

(Keller and Bustamante, Biophys. J. 2000)

slide35

Force dependence of Vmax, KM

Vmax/KM ~ constant

KM decreases with force

Vmax decreases with force

translocation coincides with release

ATP

ATP

M1

M2

ADP

Pi

M3

ADP

Pi

Translocation coincides with release

Our data is consistent with the translocation

step coinciding with the release of products of

the catalysis

Movement

step

slide37

Step size

If noise Dxrms >> step size d, we cannot measure d directly

Measure distribution of times spent in a bin of size Dl (which can be >>Dxrms and d) “residence time ”

Distribution of residence times is well-defined

For an enzyme that performs the steps in a purely random

fashion (i.e., its stepping follows Poisson’s statistics) and

has one rate-limiting step, this distribution is:

1

(Dl/d-1)!

tDl/d-1

tDl/d

P(t,Dl/d) = e-t/t, t=d/v

Dl, v are known d?

<t>=Dl/v, <t2>-<t>2=dDl/v2

slide38

Residence times

Measure residence time distributions P(t,Dl) vs. [ATP]

Fit to distributions to obtain step size d

d = 2.15

Extrapolation to [ATP]  0 gives d~2bp

current questions
Current questions

• What is the organization of the DNA inside the

capsid

• Does the motor rotate during translocation?

• How does the DNA structure affect the activity

of the motor?

- chargeless DNA

- ssDNA

• What is the molecular mechanism of energy

transduction?

slide40

“Once I met a man who grabbed a

cat by the tail and learnt 40% more

about cats that the man who didn’t”

Mark Twain