Topic 4: Equilibrium binding and chemical kinetics

Outline:

Applications, applications, applications …

• use Boltzmann to look at receptor-ligand binding

• use Boltzmann to look at PolII-DNA binding and gene transcription

• law of mass action and chemical equilibrium

• revisit receptor-ligand binding using law of mass action

• cooperative binding and switch like response

Ligand binding to a receptor

Consider the problem of a ligand binding to a receptor.

Q: What is the probability that the receptor is bound by ligand?

The answer will depend on the ligand concentration and on the energy differnce
for a ligand to be bound or unbound to the receptor
 Definining the problem

Free ligand in solution has energy, 𝜀𝑠𝑜𝑙 and when bound to receptor, 𝜀𝑏

Use the Boltzmann distribution to calculate P(bound)

Need to enumerate states - like what you did in the assignment

Ω boxes (# of states)
Using Boltzmann:

Boltzmann says that the probability of a state is: (add up all the way that state
can occur x Boltzmann weight) / (all states x Boltzmann weight)

Using results from previous slide:

Ω𝐿−1 −(𝐿−1)𝜀𝑠𝑜𝑙 /𝑘𝑇 𝐿 −Δ𝜀/𝑘𝑇

𝑒 −𝜀𝑏 /𝑘𝑇 𝑒 ( )𝑒
𝐿−1 ! Ω
𝑝𝑏𝑜𝑢𝑛𝑑 = 𝐿 =
Ω −𝐿𝜀𝑠𝑜𝑙 /𝑘𝑇 Ω 𝐿−1 𝐿
𝑒 + 𝑒 −𝜀𝑏 /𝑘𝑇 𝑒 −(𝐿−1)𝜀𝑠𝑜𝑙 /𝑘𝑇 1 + Ω 𝑒 −Δ𝜀/𝑘𝑇
𝐿! 𝐿−1 !

where Δ𝜀 = 𝜀𝑏 − 𝜀𝑠𝑜𝑙 < 0

 Equilibrium binding constant, K

We can express this result in terms

of concentration: 𝑙 = 𝐿/𝑉 and 𝑙0 = Ω/𝑉

so
Δ𝜀
𝑙 −
𝑙0
𝑒 𝑘𝑇
𝑝𝑏𝑜𝑢𝑛𝑑 = Δ𝜀
𝑙 −
1+ 𝑙0
𝑒 𝑘𝑇

or
𝑙
𝐾𝐷
𝑝𝑏𝑜𝑢𝑛𝑑 =
𝑙
1+ 𝐾
𝐷

where we have defined the equilibrium

dissociation constant, 𝐾𝐷

𝐾𝐷 = 𝑙0 𝑒 Δ𝜀/𝑘𝑇 and has units of concentration,

where if the boxes are 1 nm3, 𝑙0 = 0.6 M

Regulating Gene Expression

What proteins get made by a cell

is determined by what genes get
expressed

this is governed by transcription,

which depends on RNA polymerase
binding to the promoter regions of
genes

The first level of regulating which

proteins get made and how much is
by controlling transcription

the transcriptome is the collection of

every RNA molecule that has been
transcribed from the genome
 Statistical mechanics of gene expression

A gene can be transcribed into RNA only if the RNA polymerase (Pol II)binds to its promoter

Thus the probability that a promoter will be bound by Pol II is essential for cellular function

Controlling the probability of PolII binding to promoters (via sequence changes) is one
of the key regulatory mechanisms governing gene expression – as we will see in our
calculation that follows
 The states of PolII binding to DNA

Let’s consider that we have, P, Pol II polymerases, and they are all bound to the DNA
(this is not a horrible assumption, as there is very little free polymerase in the cytoplasm)

The DNA can be thought of as consisting of 𝑁𝑁𝑆 , non-specific binding sites

 Binding to a promoter

The non-specific binding of PolII to DNA comes mostly from attractive electrostatic
energy between the +ve charged PolII and the –ve charged DNA. This non-specific
interaction does not depend on DNA sequence.

specific binding non-specific binding

Unlike non-specific binding, specific binding depends very much on the underlying
DNA sequence. Promoters have specific DNA sequences that form hydrogen bonds
and other specific interactions with regions of PolII leading to lower binding energy
 Writing down all the terms

We will focus on the probability to bind to one specific promoter, all other sites
will be considered non-specific

This is essentially the same as the ligand – receptor problem.

 Applying the Boltzmann Distribution

Using results from previous slide:

𝑁𝑁𝑆 𝑃−1 −(𝑃−1)𝜀𝑁𝑆 /𝑘𝑇 𝑃

𝑒 −𝜀𝑠 /𝑘𝑇 𝑒 (𝑁 )𝑒 −Δ𝜀/𝑘𝑇
𝑃−1 ! 𝑁𝑆
𝑝𝑏𝑜𝑢𝑛𝑑 = =
𝐿
𝑁𝑁𝑆 −𝑃𝜀𝑁𝑆 /𝑘𝑇 𝑁 𝑃−1 𝑃
𝑒 −𝜀
+𝑒 𝑆 /𝑘𝑇 𝑁𝑆
𝑒 −(𝑃−1)𝜀𝑁𝑆 /𝑘𝑇 1 + 𝑒 −Δ𝜀/𝑘𝑇
𝑃! 𝑃−1 ! 𝑁 𝑁𝑆

where Δ𝜀 = 𝜀𝑠 − 𝜀𝑁𝑆 < 0

 Sequence controls the probability of PolII being bound

viral promoter
The probability of a promoter being bound is

Δ𝜀
𝑃 −
𝑁𝑁𝑆
𝑒 𝑘𝑇
𝑝𝑏𝑜𝑢𝑛𝑑 = Δ𝜀
𝑃 −
1+ 𝑁 𝑒 𝑘𝑇
𝑁𝑆 lactose metabolism promoter

The number of polymerases, P is

roughly a constant across cells, as is the
number of non-specific binding sites

A cell controls the probability of a promoter

being bound by controlling Δ𝜀. It does
this by changing the DNA sequence in the
promoter.

Shown are the probabilities of being bound for the lac promoter in E. coli (this controls
the machinery that processes the important sugar source, lactose), as well as the promoter
of a gene in a bacterial virus. The virus has a much stronger promoter. Here,
Δ𝜀𝑙𝑎𝑐 = −2.9kT and Δ𝜀𝑇7 = −8.1kT, with 𝑁𝑁𝑆 = 5 × 106
 Law of mass action and chemical equilibrium

We can also arrive at the previous results using results from equilibrium chemistry

Consider the reaction:

chemical equilibrium occurs when the flow from the left equals the flow from the right

or 𝑘+ 𝐴 𝐵 = 𝑘− [𝐴𝐵]

reactants product

𝐴 [𝐵] 𝑘−
or = = 𝐾𝐷 ≡ dissociation constant
[𝐴𝐵] 𝑘+

so at equilibrium, the ratio of the concentration of the reactants to the product is

the dissociation constant – it has units of concentration.

We will identify this with our previous result for the dissociation constant that depended
on the energy difference between the reacting and product states
General result for chemical reactions

In general, if we have multiple species involved in the reaction with different

numbers of each coming together to bind

𝑛𝐴 + 𝑚𝐵 + 𝑝𝐶 ⇌ 𝐷
then equilibrium occurs when

𝐴𝑛 𝐵𝑚 𝐶 𝑝 𝑛+𝑚+𝑝−1
𝐷
= 𝐾𝐷

for example consider:

2𝐴 + 𝐵 ⇌ 𝐶
then

[𝐴]2 [𝐵]
[𝐶]
= 𝐾𝐷2
Ligand-Receptor binding redux:

Let’s consider the problem of ligand binding to a receptor again. Previously we

showed that,
Δ𝜀
𝑙 −
𝑒 𝑘𝑇
𝑙0
𝑝𝑏𝑜𝑢𝑛𝑑 = Δ𝜀
𝑙 −
1+ 𝑙 𝑒 𝑘𝑇
0
Using equilibrium chemistry:

looks familiar
Ligand receptor binding experiments

𝑝 [𝐿]
𝑏𝑜𝑢𝑛𝑑=
𝐾𝐷 +[𝐿]

𝐾𝐷
𝐾𝐷

Comparing to our previous result, we see that as before, 𝐾𝐷 = 𝑙0 𝑒 Δ𝜀/𝑘𝑇 .

We also see that the dissociation constant corresponds to the concentration where
the receptor is ½ bound. The above plots show how the binding energy can be
measured experimentally.
Cooperative binding

Many binding events in molecular biology are cooperative, meaning that the binding
of multiple sites is better than having each site bound individually
cooperative interaction
between sites

In cooperative binding there is additional binding energy that occurs between the
bound ligands
Cooperative binding: Hill function

More generally, if there is cooperativity amongst, n binding sites:

[𝐿]𝑛
𝑝𝑏𝑜𝑢𝑛𝑑 = 𝑛 Hill coefficient – measures
𝐾𝐷 + [𝐿]𝑛
cooperativity

the higher the cooperativity, the more

switch like response to binding as
a function of input concentration

this can lead to chemical systems being

‘digital’. That is they can be either ‘on’
or ‘off’ as a function of input ligand.
Back to gene regulation (Ch 19)

Besides controlling the promoter sequence to regulate PolII binding, cells use
transcription factors to enhance or repress transcription

transcription factors

Transcription factors have cooperative interactions with PolII and themselves

Their binding leads to regulatory logic – i.e. various logic functions on the factors
lead to the expression of the gene. This leads to cellular computation.
Transcriptional activators:

Consider a simple system of the PolII plus an additional activator protein, A

The PolII can bind to a promoter, and ther activator to a specific site upstream
of the promoter

there is a cooperative interaction between the activator and PolII that helps
to stabilize the PolII on the promoter  enhancing the probability of being bound
and therefore more transcription
Enumerating states

Just as before, you enumerate

the possible configurations

there’s a lot more to keep track of

fortunately we can also treat this

problem using equilibrium chemistry

you’ll work this out on an assignment

 Summary:

• We’ve begun to apply the Boltzmann distribution to calculate probabilities

• We saw how we can define the strength of binding in terms of the

energy gained in binding

• gene transcription depends critically on the probability of PolII binding

to the promoter

• we can treat binding reactions using equilibrium chemistry

• cooperative binding leads to switch like responses as a function of

chemical input

• transcriptional logic is implemented by cooperative interactions between

transcription factors and PolII