Expression of many genes in cells Expression of many genes in cells

are regulatedare 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.

Gene Regulation in ProkaryotesGene Regulation in Prokaryotes

Principles of Transcription Principles of Transcription

RegulationRegulation

� What are the regulatory proteins?

� 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 Gene Expression is Controlled by

Regulatory ProteinsRegulatory Proteins

�� Gene expression is very often controlled by Gene expression is very often controlled by ExtracellularExtracellular Signals, which are mediated by Signals, which are mediated by regulatory proteinsregulatory proteins::

�� PositivePositive regulators or activators regulators or activators INCREASEINCREASE the transcriptionthe transcription

�� NegativeNegative regulators or repressors regulators or repressors

DECREASE DECREASE or or ELIMINATE ELIMINATE the transcriptionthe transcription

Most activators and repressors act at Most activators and repressors act at

the level of transcription initiation 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.

� 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.

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 activator and repressor, RNA polymerase occasionally binds the promoter spontaneously and initiates low level transcription.

� Binding of repressors block transcription

� Recruitment of RNA polymerase by activator gives high levels of transcription

Targeting transition to the open Targeting transition to the open

complex:complex:AllosteryAllostery regulationregulation

(after the RNA polymerase Binding)(after the RNA polymerase Binding)

In some cases, RNAP binds the promoters efficiently, but does not spontaneously lead to the open complex, resulting in no or low transcription.

Some activators can bind to the closed complex, inducing conformational change in either RNAP or DNA promoter, which converts the closed complex to open complex and thus promotes the transcription. This is an example of allostery regulation.

AllosteryAllostery regulationregulation

Repressors Repressors

CCan work in ways:(1) blocking the promoter binding. (2) blocking the transition to the open

complex.(3) blocking promoter escape

� The regulator proteins can function even binding at a DNA site far away from the promoter region, through protein-protein interaction and DNA looping.

Action at a Distance and DNA Looping.

Cooperative binding of

proteins to adjacent sites

Cooperative binding of

proteins at two separate

sites

Cooperative bindingCooperative binding (recruitment) (recruitment)

and and allosteryallostery have many roles in have many roles in

gene regulationgene 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 turn on a gene expression.

Regulation of Transcription Regulation of Transcription

Initiation : Initiation :

Examples from BacteriaExamples from Bacteria

LacLac operonoperonThe lactose Operon

Point 1: Composition of the Lac operon

1. Lactose operon contains 3 structural genes and 2 control elements.

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 (polycistronicpolycistronic mRNAmRNA) under the control of a single promoter.

An activator and a repressor together An activator and a repressor together

control the control the LacLac operonoperon expressionexpression

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

The activity of The activity of LacLac repressor and CAP repressor and CAP

are controlled are controlled allostericallyallosterically by signals.by signals.

Lactose is converted to allolactose by ββββ-galactosidase, therefore lactose can indirectly turn off the repressor.

Glucose lowers the cellular cAMP level, therefore, glucose indirectly turn off CAP.

Allolactose binding: turn off Lac repressor

cAMP binding: turn on CAP

ii pp oo zz yy aa

Very low level of lac mRNAVery low level of lac mRNA

Absence of lactoseAbsence of lactose

ActiveActive

i p o z y a

β-Galactosidase

Permease

Transacetylase

Presence of lactose

Inactive

Lack of inducer: the lacrepressor block all but a very low level of transcription of lacZYA .

When Lactose is present, the low basal level of permease allows its uptake, and β-galactosidasecatalyzes the conversion of some lactose to allolactose.

Allolactose acts as an inducer, binding to the lacrepressor and inactivate it.

Response to lactose

Response to glucose: CRPThe mechanism of the binding of The mechanism of the binding of

regulatory proteins to their sitesregulatory proteins to their sitesCAP and CAP and LacLac repressor have opposing effects on RNA repressor have opposing effects on RNA polymerase binding to the promoterpolymerase binding to the promoter

Repressor binding physically prevents RNAP from Repressor binding physically prevents RNAP from binding to the promoter.binding to the promoter.

CAP binds to a site upstream of the promoter, and helps CAP binds to a site upstream of the promoter, and helps RNA polymerase binds to the promoter by physically RNA polymerase binds to the promoter by physically interacting with RNAP. This cooperative binding interacting with RNAP. This cooperative binding stabilizes the binding of polymerase to stabilizes the binding of polymerase to PPlaclac. .

Gene Regulation in EukaryotesGene Regulation in Eukaryotes

Similarity of regulation between eukaryotes and prokaryote

1.Principles are the same:

• signals (信号信号信号信号), • 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.

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.

A lot more regulator bindings sites in A lot more regulator bindings sites in

multicellularmulticellular organisms reflects the organisms reflects the

more extensive signal integration more extensive signal integration

� 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 at a distance is much more common in eukaryotes.

� Insulators or boundary elements are regulatory sequences between enhancers and promoters. They

block activation of a linked promoter by activator bound at the enhancer, and therefore ensure activators work discriminately.

Conserved Mechanisms of Conserved Mechanisms of

Transcriptional Regulation Transcriptional Regulation

from Yeast to Mammalsfrom Yeast to Mammals

� 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.

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Eukaryotic activators have separate DNA binding and activating functions. Both are very often on

separate domains of the protein.*****

Gal4 bound to its site on DNA

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

� Gal4 is the most studied eukaryotic activator

� Gal4 activates transcription of the galactosegenes in the yeast S. cerevisae.

� Gal4 binds to four sites (UASG) upstream of GAL1,

and activates transcription 1,000-fold in the presence of galactose

The regulatory sequences of the Yeast GAL1 gene

�� Eukaryotic activators also work similarly as Eukaryotic activators also work similarly as

bacteria, but recruit polymerase indirectly bacteria, but recruit polymerase indirectly

in two ways:in two ways:

�� 1. Interacting with parts of the 1. Interacting with parts of the transcription transcription

machinerymachinery..

�� 2. 2. Recruiting Recruiting nucleosomenucleosome modifiersmodifiers that that

alter chromatin in the vicinity of a gene.alter chromatin in the vicinity of a gene.

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes, such as the Mediator and the TFⅡⅡⅡⅡD complex. Activators interact with one or more of these complexes and recruit them to the gene.

Activators also recruit modifiers that Activators also recruit modifiers that

help the transcription machinery bind help the transcription machinery bind

at the promoterat the promoter

Two types of Two types of NucleosomeNucleosome modifiers :modifiers :

Those add chemical groups to the tails of Those add chemical groups to the tails of

histoneshistones, , such as such as histonehistone acetyl acetyl transferasestransferases

((HATsHATs))

Those remodel the Those remodel the nucleosomesnucleosomes, , such as the such as the

ATPATP--dependent activity of SWI/SNF.dependent activity of SWI/SNF.

How the How the nucleosomenucleosome modification help modification help activate a gene?*****activate a gene?*****

1.1. ““LoosenLoosen”” the chromatin structurethe chromatin structure by by chromosome remodeling and chromosome remodeling and histonehistonemodification such as modification such as acetylationacetylation, which , which uncover DNAuncover DNA--binding sites that would binding sites that would otherwise remain inaccessible within the otherwise remain inaccessible within the nucleosomenucleosome..

2. Adding acetyl groups to Adding acetyl groups to histoneshistones helps the helps the binding of the transcriptional machinerybinding of the transcriptional machinery. . Therefore, a gene bearing acetylated Therefore, a gene bearing acetylated nucleosomesnucleosomes at its promoter have a higher at its promoter have a higher affinity for the transcriptional machinery than affinity for the transcriptional machinery than the one with the one with unacetylatedunacetylated nucleosomesnucleosomes. . Local alterations in chromatin directed by

activators

Many enkaryotic activators－－－－particularlyin higher eukaryotes－－－－work from a distance.How?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.

3. Action at a distance: loops and 3. Action at a distance: loops and

insulatorsinsulators

Specific Specific ciscis--acting elements called acting elements called insulatorsinsulators

control the actions of activators, preventing the control the actions of activators, preventing the

activating the nonactivating the non--specific genesspecific genes

Transcriptional SilencingTranscriptional Silencing

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 [histonedeacetylases removing the acetyl groups].

Some modifier adds methyl groups to the histone tails, which frequently repress the transcription [histonemethyltransferase].This modification causes transcriptional silencing.

Gene Gene ““SilencingSilencing”” by Modification of by Modification of

HistonesHistones and DNAand 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 Transcriptional silencing is associated with

modification of modification of nucleosomesnucleosomes that alters the that alters the

accessibility of a gene to the transcriptional machinery accessibility of a gene to the transcriptional machinery

and other regulatory proteins. and other regulatory proteins.

�� The modification enzymes for silencing include The modification enzymes for silencing include

deacetylasesdeacetylases, DNA , DNA methylasesmethylases..

�� Transcription can also be silenced by Transcription can also be silenced by methylationmethylation of of

DNA by DNA by histonehistone methyltransferasemethyltransferase. This enzyme have . This enzyme have

been recently found in yeast, but is common in been recently found in yeast, but is common in

mammalian cells. mammalian cells.

�� In higher eukaryotes, silencing is typically associated In higher eukaryotes, silencing is typically associated

with chromatin containing with chromatin containing histoneshistones that both that both

deacetylateddeacetylated and and methylatedmethylated..

Three other ways in which an eukaryotic repressor works include:

� Competes with the activator for an overlapped binding site.

� Binds to a site different from that of the activator, but physically interacts with an activator and thus block its activating region.

� Binds to a site upstream of the promoter, physically interacts with the transcription machinery at the promoter to inhibit transcription initiation.

Competes for the activator binding

Inhibits the function of the activator.

Ways in which eukaryotic

repressor work******

Binds to the transcription machinery

Recruits nucleosomemodifiers (most

common******)

Signal Integration and Combinatorial Signal Integration and Combinatorial

ControlControl

1.1. Activators work together Activators work together synergisticallysynergistically to to

integrate signals.integrate signals.

In In multicellularmulticellular organisms, signal integration is organisms, signal integration is

used extensively. In some cases, numerous used extensively. In some cases, numerous

signals are required to switch a gene on. signals are required to switch a gene on.

However, each signal is transmitted to the However, each signal is transmitted to the

gene by a separate regulator, and therefore, gene by a separate regulator, and therefore,

multiple activators often work together, and multiple activators often work together, and

they do so synergistically (two activators they do so synergistically (two activators

working togetherworking together is greater than the sum of is greater than the sum of

each of them working alone).each of them working alone).

45

Figure 17-14: Cooperative binding of activators

Figure 17-14: Cooperative binding of activators

a.“Classical”cooperative binding.

b. Both proteins interacting with a third protein.

c. The first protein recruit a nucleosomeremodeller whose action reveal a binding site for the second protein.

d. Binding a protein unwinds the DNA from nucleosome a little, revealing the binding site for another protein.

Signal Transduction and the Control of Signal Transduction and the Control of

Transcriptional RegulatorsTranscriptional Regulators

1. Signals are often communicatedcommunicated to

transcriptional regulators through signal transduction pathway

Environmental Signals/Information 1. Small molecules such as sugar, histamine.2. Proteins released by one cell and received by

another.

In eukaryotic cells, most signals are communicated to genes through signal transduction pathway (indirect), in which the initiating ligand is detected by a specific cell surface receptor.

3*. The signal is then relayed to the relevanttranscriptional regulator.transcriptional regulator.

Signal transduction pathway***Signal transduction pathway***

1. The initial ligand (“signal”) binds to anextracellularextracellular domaindomain of a specific cell surface receptor

2. The signal is thus communicated to theintracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4. The transcriptional regulator control thetarget gene expressiontarget gene expression.

Signal transduction Signal transduction

pathway***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).