Expression of many genes in cells

are regulated
Housekeeping genes: expressed constitutively,
essential for basic processes involving in cell
replication and growth.

Inducible genes: expressed only when they

are activated by inducers or cellular factors.

Principles of Transcription
Regulation

What are the regulatory proteins?

Gene Regulation in Prokaryotes
Which steps of gene expression to be targeted?

How to regulate? (recruitment, allostery,
blocking, action at a distance, cooperative
binding)
 Gene Expression is Controlled by
Regulatory Proteins

Gene expression is very often controlled by
Extracellular Signals, which are mediated by
regulatory proteins:
proteins:

Positive regulators or activators
INCREASE the transcription

Negative regulators or repressors
DECREASE or ELIMINATE the transcription

Regulation also occurs at all stages after
transcription initiation. Why?

Allows more inputs and multiple checkpoints.

The regulation at later stages allow a
quicker response.
Most activators and repressors act at
the level of transcription initiation

Why’s that?

Transcription initiation is the most
energetically efficient step to regulate. [A wise
decision at the beginning]

Regulation at this step is easier to do well than
regulation of the translation initiation.
Targeting promoter binding: Many promoters
are regulated by activators that help RNAP
bind DNA (recruitment) and by repressors that
block the binding.
Generally, RNAP binds many promoters weakly.
Activators contain two binding sites to bind a
DNA sequence and RNAP simultaneously, can
therefore enhance the RNAP affinity with the
promoters and increases gene transcription. This is
called recruitment regulation (Cooperative binding).
On the contrary, Repressors can bind to the
operator inside of the promoter region, which
prevents RNAP binding and the transcription of the
target gene.

In absence of both Targeting transition to the open
activator and complex:
repressor, RNA
polymerase
Allostery regulation
occasionally binds the (after the RNA polymerase Binding)
promoter
spontaneously and In some cases, RNAP binds the promoters efficiently,
initiates low level but does not spontaneously lead to the open complex,
transcription. resulting in no or low transcription.

Binding of repressors Some activators can bind to the closed complex,

block transcription inducing conformational change in either RNAP or
DNA promoter, which converts the closed complex to
open complex and thus promotes the transcription.

Recruitment of RNA This is an example of allostery regulation.
polymerase by
activator gives high
levels of transcription

Allostery regulation

Repressors

Can work in ways:

(1) blocking the promoter binding.
(2) blocking the transition to the open
complex.
(3) blocking promoter escape
 Action at a Distance and DNA Looping.

The regulator proteins can function even binding
at a DNA site far away from the promoter region,
through protein-protein interaction and DNA
looping.
Cooperative binding of
proteins to adjacent sites
Cooperative binding (recruitment)
and allostery have many roles in
gene regulation
For example: group of regulators often
bind DNA cooperatively (activators
and/or repressors interact with each
other and with the DNA, helping each
other to bind near a gene they
regulated).

To produce sensitive switches to rapidly

Cooperative binding of
proteins at two separate
turn on a gene expression.
sites

Lac operon
The lactose Operon
Point 1: Composition of the Lac operon
1. Lactose operon contains 3 structural genes and 2
Regulation of Transcription control elements.

Initiation :
Examples from Bacteria
The enzymes encoded by lacZ, lacY, lacA are required
for the use of lactose as a carbon source. These genes
are only transcribed at a high level when lactose is
available as the sole carbon source.
The lacZ, lacY, lacA genes are transcribed into a
single lacZYA mRNA (polycistronic
polycistronic mRNA)
mRNA under the
control of a single promoter.
An activator and a repressor together The activity of Lac repressor and CAP
control the Lac operon expression are controlled allosterically by signals.

Allolactose binding: turn off Lac repressor

The activator: CAP (Catabolite Activator
Protein) or CRP (cAMP Receptor
Protein,cAMP); responses to the glucose level.
The repressor: lac repressor that is encoded
by LacI gene; responses to the lactose.
Sugar switch-off mechanism
cAMP binding: turn on CAP
Lactose is converted to allolactose by β-
galactosidase, therefore lactose can indirectly turn
off the repressor.
Glucose lowers the cellular cAMP level, therefore,
glucose indirectly turn off CAP.

Response to lactose Absence of lactose

Lack of inducer: the lac
repressor block all but a ii pp oo zz yy aa

very low level of Active

Active
transcription of lacZYA .
Very
Very low
low level
level of
of lac
lac mRNA
mRNA

When Lactose is present,

the low basal level of Presence of lactose
permease allows its uptake,
i p o z y a
and β-galactosidase
catalyzes the conversion of
some lactose to allolactose.
Allolactose acts as an Inactive
inducer, binding to the lac
repressor and inactivate it. Permease
β-Galactosidase Transacetylase
 The mechanism of the binding of
Response to glucose: CRP regulatory proteins to their sites
CAP and Lac repressor have opposing effects on RNA
polymerase binding to the promoter

Repressor binding physically prevents RNAP from

binding to the promoter.

CAP binds to a site upstream of the promoter, and helps

RNA polymerase binds to the promoter by physically
interacting with RNAP. This cooperative binding
stabilizes the binding of polymerase to Plac.

Similarity of regulation between

eukaryotes and prokaryote

1.Principles are the same:

• signals ( 信号 ),
Gene Regulation in Eukaryotes • activators and repressors
• recruitment and allostery, cooperative binding

2. The gene expression steps subjected to

regulation are similar, and the initiation of
transcription is the most pervasively regulated
step.
 Difference in regulation between
eukaryotes and prokaryote*****
1. Pre-mRNA splicing adds an important step for
regulation.
A lot more regulator bindings sites in
multicellular organisms reflects the
more extensive signal integration

2. The eukaryotic transcriptional machinery is

more elaborate than its bacterial counterpart.

3. Nucleosomes and their modifiers influence

access to genes.

4. Many eukaryotic genes have more regulatory

binding sites and are controlled by more
regulatory proteins than are bacterial genes.

Enhancer : a given site binds regulator responsible

for activating the gene. Alternative enhancer binds
different groups of regulators and control
expression of the same gene at different times and
places in responsible to different signals. Activation
Conserved Mechanisms of
at a distance is much more common in eukaryotes. Transcriptional Regulation

Insulators or boundary elements are regulatory from Yeast to Mammals
sequences between enhancers and promoters. They
block activation of a linked promoter by activator
bound at the enhancer, and therefore ensure
activators work discriminately.
 Eukaryotic activators have separate DNA binding
and activating functions. Both are very often on
separate domains of the protein.*****

The basic features of gene regulation are the
same in all eukaryotes, because of the similarity
in their transcription and nucleosome structure.

The typical eukaryotic activators works in a

manner similar to the simplest bacterial case.

Repressors work in a variety of ways.

Gal4 bound to its site on DNA 30

Eukaryotic activators---Example 1: Gal4*****

Gal4 is the most studied eukaryotic activator

Gal4 activates transcription of the galactose
Eukaryotic activators also work similarly as
genes in the yeast S. cerevisae. bacteria, but recruit polymerase indirectly

Gal4 binds to four sites (UASG) upstream of GAL1, in two ways:
and activates transcription 1,000-fold in the
presence of galactose
1. Interacting with parts of the transcription
machinery.
machinery.

2. Recruiting nucleosome modifiers that

alter chromatin in the vicinity of a gene.

The regulatory sequences of the Yeast GAL1 gene

The eukaryotic transcriptional machinery contains
polymerase and numerous proteins being organized
to several complexes, such as the Mediator and
Ⅱ
the TF D complex. Activators interact with one
or more of these complexes and recruit them to
the gene.
Activators also recruit modifiers that
help the transcription machinery bind
at the promoter
Two types of Nucleosome modifiers :
Those add chemical groups to the tails of
histones,
histones, such as histone acetyl transferases
(HATs)
HATs)
Those remodel the nucleosomes,
nucleosomes, such as the
ATP-
ATP-dependent activity of SWI/SNF.

How the nucleosome modification help

activate a gene?*****
1. “Loosen”
Loosen” the chromatin structure by
chromosome remodeling and histone
modification such as acetylation,
acetylation, which
uncover DNA-
DNA-binding sites that would
otherwise remain inaccessible within the
nucleosome.
nucleosome.
2. Adding acetyl groups to histones helps the
binding of the transcriptional machinery.
machinery.
Therefore, a gene bearing acetylated
nucleosomes at its promoter have a higher
affinity for the transcriptional machinery than
the one with unacetylated nucleosomes.
nucleosomes. Local alterations in chromatin directed by
activators
3. Action at a distance: loops and Specific cis-
cis-acting elements called insulators
control the actions of activators, preventing the
insulators
－
activating the non-
non-specific genes
Many enkaryotic activators particularly
－
in higher eukaryotes work from a distance.
How?
1. Some proteins help, for example Chip protein in
Drosophila.
2. The compacted chromosome structure help.
DNA is wrapped in nucleosomes in eukaryotes.
So sites separated by many base pairs may not
be as far apart in the cell as thought.

Transcriptional Silencing Gene “Silencing” by Modification of

Transcriptional Silencing is a specialized form of repression
that can spread along chromatin, switching off multiple
genes without the need for each to bear binding sites for
specific repressor.
In eukaryotes, most repressors do not repress transcription
by binding to sites that overlap with the promoter and thus
block binding of polymerase. (Bacteria often do so)
Commonly, eukaryotic repressors recruit nucleosome modifiers
that compact the nucleosome or remove the groups
recognized by the transcriptional machinery [histone
deacetylases removing the acetyl groups].
Some modifier adds methyl groups to the histone tails, which
frequently repress the transcription [histone
methyltransferase].This modification causes transcriptional
silencing.
Histones and DNA

Transcriptional silencing is a position effect.
(1) A gene is silenced because of where it is located,
not in response to a specific environmental signal.
(2) Silencing can spread over large stretches of DNA,
switching off multiple genes, even those quite
distant from the initiating event.

The most common form of silencing is associated
with a dense form of chromatin called
“heterochromatin”.

Heterochromatin is frequently associated with
particular regions of the chromosome, notably the
telomeres, and the centromeres.

In mammalian cells, about 50% of the genome is
estimated to be in some form of heterochromatin.

Transcriptional silencing is associated with
modification of nucleosomes that alters the Three other ways in which an eukaryotic repressor
accessibility of a gene to the transcriptional machinery works include:
and other regulatory proteins.

Competes with the activator for an overlapped

The modification enzymes for silencing include binding site.
deacetylases,
deacetylases, DNA methylases.
methylases.

Binds to a site different from that of the

Transcription can also be silenced by methylation of activator, but physically interacts with an
DNA by histone methyltransferase.
methyltransferase. This enzyme have activator and thus block its activating region.
been recently found in yeast, but is common in
mammalian cells.
Binds to a site upstream of the promoter,
physically interacts with the transcription

In higher eukaryotes, silencing is typically associated machinery at the promoter to inhibit
with chromatin containing histones that both transcription initiation.
deacetylated and methylated.
methylated.

Ways in which Signal Integration and Combinatorial

eukaryotic Control
repressor work***
***
1. Activators work together synergistically to
Competes for the integrate signals.
activator binding
In multicellular organisms, signal integration is
Inhibits the used extensively. In some cases, numerous
function of the signals are required to switch a gene on.
activator. However, each signal is transmitted to the
gene by a separate regulator, and therefore,
Binds to the multiple activators often work together, and
transcription they do so synergistically (two activators
machinery working together is greater than the sum of
each of them working alone).
Recruits nucleosome
modifiers (most
common******)
 a.“Classical”
cooperative binding. Signal Transduction and the Control of
b. Both proteins Transcriptional Regulators
interacting with a
third protein. 1. Signals are often communicated to
transcriptional regulators through signal
c. The first protein
transduction pathway
recruit a nucleosome
remodeller whose
Environmental Signals/Information
action reveal a
1. Small molecules such as sugar, histamine.
binding site for the
2. Proteins released by one cell and received by
second protein.
another.
d. Binding a protein
unwinds the DNA In eukaryotic cells, most signals are communicated
from nucleosome a to genes through signal transduction pathway
little, revealing the (indirect), in which the initiating ligand is detected
Figure 17-14: binding site for by a specific cell surface receptor.
Cooperative binding another protein.
of activators 45

Signal transduction pathway***

1. The initial ligand (“signal”) binds to an
extracellular domain of a specific cell surface
receptor

2. The signal is thus communicated to the

intracellular domain of receptor (via an
allosteric change or dimerization )

3*. The signal is then relayed to the relevant

transcriptional regulator.

4. The transcriptional regulator control the Signal transduction

target gene expression.
expression pathway***
Signals control the activities of eukaryotic
transcriptional regulators in a variety of ways.
Mechanism 1: unmasking an activating region.
A conformational change to reveal the previously buried
activating region.
Mechanism 2: Transport into and
out of the nucleus .
When not active, many
activators and repressors are
held in the cytoplasm. The
signalling ligand causes them
to move into the nucleus
where they activate
transcription.

Other Mechanisms #1: A cascade
of kinases that ultimately cause
the phosphorylation of regulator
in nucleus (new).

Other Mechanisms #2: The
activated receptor is cleaved by
cellular proteases, and the c-
terminal portion of the receptor
enters the nuclease and activates
the regulator (new).