Review Lecture 2

Michaelis-Menten kinetics

k1 k2
E + S ES E + P
k-1

d[S]
= −k 1[E][S] + k −1 [ES]
dt
d[E]
= −k1 [E][S] + (k −1 + k 2)[ES]
dt
d[ES]
= k1 [E][S] − (k −1 + k 2)[ES]
dt
dP
= k 2[ES] ≡ v
dt
 Eo = [E] + [ES]

d[S]
= −k 1Eo [S] + (k 1[S] + k −1 )[ES]
dt
d[ES]
= k1Eo[S] − (k1[S] + k −1 + k 2)[ES]
dt

Initial conditions:

[S]t=0 = So
[E]t=0 = Eo
[ES]t=0 = 0
[P]t=0 = 0
 v maxSo
v0 =
K m + So
Good approximation if So >> Eo

in this case S0 ~ [S] at the start of

quasi-steady state
 Review Lecture 2
Equilibrium binding and cooperativity

S+P ↔P
j− 1 j

Adair’s Equation:
K [S]+ 2K K [S]2 + 3K K K [S]3 +...+ nK K ...Kn[S]n
r= 1 1 2 1 2 3 1 2
1 + K [S]+ K K [S]2 +...+ K K ...Kn[S]n
1 1 2 1 2
[P ]
j macroscopic association constant
K =
j [P ][S] for transitions between state j-1 and j
j− 1
 Note #1 Detailed balance

k+1 k+2 k+n

Po P1 P2 ... Pn-1 Pn
k-1 k-2 k-n

d[P ]
0= o = −k [P ][S]+ k [P ]
dt +1 o −1 1
d[P ]
0= 1 = −k [P ][S]+ k [P ]+ k [P ][S]− k [P ]=
dt +2 1 −2 2 +1 o −1 1
= −k [P ][S]+ k [P ]
+2 1 −2 2

etc. k [P ]
+j j
K ≡ =
j k [P ][S]
−j j− 1
I Identical and independent binding sites

k+ S k+

k- k-
S S
k+ k+

k- S k-

K=k+/k- K1=2K K2=K/2

2K[S]+ 2K2[S]2 2K[S]

use Adair: r = =
1 + 2K[S]+ K2[S]2 1 + K[S]
II Non-identical and independent binding sites

k+ S k+*

k- k-*
S S
k+* k+
k-* S k-

K=k+/k-
K*=k+*/k-*

K[S] K*[S]
Independent binding: r = +
1 + K[S] 1 + K*[S]
III Identical and interacting binding sites

k+ S k+*

k- k-*
S S
k+ k+*
k- S k-*

K=k+/k- K1=2K K2=K*/2

K*=k+*/k-*
2K[S]+ 2KK*[S]2
use Adair: r=
1 + 2K[S]+ KK*[S]2
Cooperativity

2K[S]+ 2KK*[S]2
r=
1 + 2K[S]+ KK*[S]2
x(1 + βx)
Y=
β = K*/K
1 + 2x + βx2
x = K[S]

β > 1: positive cooperativity

β > 2: sigmoidal curve
β < 1: negative cooperativity
(always: d2Y/dx2 < 0)
Hill number for ‘real’ dimer
Introduction phage biology

Phage genome:
48512 base pairs ~ 12 kB
‘phage.jpg’ ~ 10 kB

Image removed due to copyright considerations.

See Ptashne, Mark. A genetic switch: phage lambda.
3rd ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2004.
The lysis-lysogeny decision:

As the phage genome is injected

phage genes are transcribed and
translated by using the host’s
machinery.

Which set of phage proteins are

expressed determines the fate of the
phage: lysis or lysogeny

Image by MIT OCW.

Image removed due to copyright considerations.

A lysogen is immune to
invasion of another phage.
Repressor dimers turn off genes
in the injected phage
chromosome. High concentration
of repressor keeps cell in
lysogenic state.
 The lysis-lysogeny decision is a genetic switch

only ‘space’ for one RNA polymerase (mutual exclusion)

Image by MIT OCW. After Ptashne, Mark. A genetic switch : phage lambda. 3rd ed. Cold Spring Harbor, N.Y. :
Cold Spring Harbor Laboratory Press, 2004.
 Single repressor dimer bound - three cases:

I Negative control, dimer binding to OR2 inhibits

RNAp binding to right PR promoter.

Positive control, dimer binding to OR2 enhances

RNAp binding to left PRM promoter.

Image removed due to copyright considerations.

See Ptashne, Mark. A genetic switch: phage lambda.
3rd ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2004.
II Negative control, dimer binding to OR1 inhibits
RNAp binding to right PR promoter.

Negative control, dimer binding to OR1 inhibits

RNAp binding to left PRM promoter (too distant).

Image removed due to copyright considerations.

See Ptashne, Mark. A genetic switch: phage lambda.
3rd ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2004.
III Negative control, dimer binding to OR3 inhibits
RNAp binding to left PRM promoter.

Positive control, dimer binding to OR3 allows

RNAp binding to right PR promoter.

Image removed due to copyright considerations.

See Ptashne, Mark. A genetic switch: phage lambda.
3rd ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2004.
 Repressor-DNA binding is highly cooperative

intrinsic association constants:

KOR1 ~ 10 KOR2 ~ 10 KOR3

However KOR2* >> KOR2 (positive cooperativity)

Image removed due to copyright considerations.

See Ptashne, Mark. A genetic switch: phage lambda.
3rd ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2004.
Flipping the switch by UV:

Image removed due to copyright considerations.

See Ptashne, Mark. A genetic switch: phage lambda.
3rd ed. Cold Spring Harbor, N.Y.:
Cold Spring Harbor Laboratory Press, 2004.

In lysogenic state, [repressor]

is maintained at constant level
by negative feedback

Image by MIT OCW.

 Repressor-DNA binding is highly cooperative

intrinsic association constants:

KOR1 ~ 10 KOR2 ~ 10 KOR3

However KOR2* >> KOR2 (positive cooperativity)

Image removed due to copyright considerations.

See Ptashne, Mark. A genetic switch: phage lambda.
3rd ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2004.
Cro dimers bind non-cooperatively to OR sites
KOR3 ~ 10 KOR2 ~ 10 KOR1

Note for repressor:

KOR1 ~ 10 KOR2 ~ 10 KOR3

Image removed due to copyright considerations.

See Ptashne, Mark. A genetic switch: phage lambda.
3rd ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2004.
Image removed due to copyright considerations.
See Ptashne, Mark. A genetic switch: phage lambda.
3rd ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2004.
 Cooperative effects make sharp switch
(‘well defined’ decision)

99.7% repression 1.0 nH=3, positively cooperative

promoter controlled by a
% Repression

single repressor-operator system 0.8

100

0.6 nH=1, non cooperative

Y
λPD 0.4

50

0.2

lysogen
0.0

0 1 2 3 4 5

Repressor concentration [S] (mM)

Images by MIT OCW.

Note: several layers of cooperativity:

dimerization, cooperative repressor binding
 How to create a mathematical model that
captures the essence of the switch ?

Images removed due to copyright considerations. See Arkin, A., J. Ross, and H. H. McAdams.
"Stochastic kinetic analysis of developmental pathway bifurcation in phage lambda-infected Escherichia coli cells."
Genetics 149, no. 4 (Aug, 1998): 1633-48.