Lecture #11

Prokaryotic and Eukaryotic transcriptional regulation and epigenetics

Coupled transcription / translation

The phenomena in bacteria where translation of the mRNA occurs simultaneously with its transcription

Operon

A unit of bacterial gene expression and regulation, including structurals genes and control elements in DNA recognized by regulator gene product(s).

trans-acting

A product that can function on any copy of its target DNA. This implies that it is a diffusible protein or RNA.

cis-acting

A site that affects the activity only of sequences on its own molecule of DNA (or RNA); this property usually implies that the site does not code for protein

regulator gene

a gene that encodes a product (typically) protein) that controls the expression of other genes (usually at the level of transcription)

structural gene

a gene that encodes any RNA or protein product other than a regulator

A regulator gene codes for

a protein that acts at a target site on DNA

• Regulator gene → mRNA → Regulator protein → Repressor protein on Target site of a Structural gene

In negative regulation

a repressor protein binds to an operator to prevent a gene from being expressed

In negative control, a trans-acting repressor binds to the

cis-acting operator to turn off transcription

In positive regulation

a transcription factor is required to bind at the promoter in order to enable RNA polymerase to initiate transcription

In positive control, a trans-acting factor must bind to

cis-acting site in order for RNA polymerase to initiate transcription at the promoter

In inducible regulation

the gene is regulated by the presence of its substrate (the inducer)

In repressible regulation

the gene is regulated by the product of its enzyme pathway (the corepressor)

Gene regulation in vivo can utilize any of these mechanisms, resulting in all four combinations:

• negative inducible

• negative repressible

• positive inducible

• positive repressible

Regulatory circuits can be designed from

all possible combinations of positive and negative control with inducible and repressible control

structural gene clusters are

coordinately controlled

Genes coding for proteins that function in the same pathway may be located adjacent to one another and controlled as a single unit that is transcribed into a

polycistronic mRNA

The lac operon occupies

~6000 bp of DNA

The lac operon is

negative inducible

Transcription of the lacZYA operon is controlled by a

repressor protein (the lac repressor) that binds to an operator that overlaps the promoter at the start of the cluster

Constitutive expression

A state in which a gene is expressed continuously

In the absence of B-galactosides, the lac operon is

expressed only at a very low (basal) level

The lac repressor and RNA polymerase bind at

sites that overlap around the transcription start point of the lac operon

The repressor protein is a

tetramer of identical subunits coded by the lac I gene

B-galacoside sugars, the substrates of the lac operon, are its

inducer

Addition of specific B-galactosides induces transcription of

all three genes of the lac operon

The lac mRNA is extremely unstable; as a result, induction can be

rapidly reversed

The lac repressor is controlled by a

small-molecule inducer

An inducer functions by converting the

repressor protein into a form with lower operator affinity

The lac repressor has

two binding sites

• one for the operator DNA

• one for the inducer

Gratuitous inducer

inducers that resemble authentic inducers of transcription, but are not substrates for the induced enzymes

The lac repressor is inactivated by an

allosteric interaction, in which binding of the inducer at its site changes the properties of the DNA-binding site (allosteric control)

The true inducer is

allolactose, not the actual substrate of B-galactosidase

Addition of the inducer converts the repressor to a

form with low affinity for the operator. THis allows RNA polymerase to initiate transcription

cis-acting constitutive mutations identify

the operator

Mutations in the operator cause

constitutive expression of all three lac structural genes

• are cis-acting and affect only those genes on the contiguous stretch of DNA

Mutations in the promoter prevent expression of lacZYA and are

uninducible and cis-acting

cis-dominant

A site or mutation that affects the properties only of its own molecule of DNA, often indicating that a site does not code for a diffusible product

Operator mutations are constitutive because the

operator is unable to bind the repressor protein

trans-acting mutations identify the

regulator gene

Mutations in the lac I gene are trans-acting and affect expression of

all lacZYA clusters in the bacterium

Mutations that eliminate lac I functions cause

constitutive expression and are recessive (lac I-)

Mutations in the DNA-binding site of the repressor are

constitutive because the repressor cannot bind the operator

Mutations that inactivate the lac I gene cause

the operon to be constitutively expressed

lac repressor binding to the operator is regulated by an

allosteric change in conformation

binding of the inducer causes a

change in the conformation of the repressor that reduces its affinity for DNA and releases it from the operator

The inducer changes the structure of the

core

The operator competes with

low-affinity sites to bind repressor

proteins that have a high affinity for a specific DNA sequence also have a

low affinity for other DNA sequences

Every base pair in the bacterial genome is the start of a low-affinity binding site for

repressor

The lac repressor binds strongly and specifically to its operator, but its released by the

inducer. All equilibrium constants are in M-1.

The large number of low-affinity sites ensures that all repressor protein is

bound to DNA

Repressor binds to the operator by moving from a low affinity site rather than by

equilibrating from solution

Virtually all the repressor in the cell is bound to

DNA

The lac operon has a second layer of control

catabolite repression

Catabolite repression

the ability of glucose to prevent the expression of a number of genes

In bacteria, catabolite repression is a

positive control system

• In eukaryotes, catabolite repression is completely different

Catabolite repressor protein (CRP)

is an activator protein that binds to a target sequence at a promoter

A small molecule inducer, cAMP, converts an activator proteins, CRP, to a form that binds the

promoter and assists RNA polymerase in initiating transcription

A dimer of CRP is activated by a single molecule of

cyclic AMP (cAMP)

cAMP is controlled by the

level of glucose in the cell

• a low glucose level allows cAMP to be made

CRP interacts with the C-terminal domain of the alpha subunit of RNA polymerase to

activate it

By reducing the level of cyclic AMP, glucose

inhibits the transcription of operons that require CRP activity

The trp Operon is a

repressible operon with three transcription units

The trp operon is negatively controlled by

the level of its product, the amino acid tryptophan (autoregulation)

The amino acid tryptophan activates an

inactive repressor encoded by trpR

A repressor (or activator) will act on

all loci that have a copy of its target operator sequence

Operators may lie at

various positions relative to the promoter

attenuation

the regulations of bacterial operons by controlling termination of transcription at a site located before the first structural gene

Termination can be controlled via

changes in RNA secondary structure that are determined by ribosome movement

THe trp operon is controlled by

attenuation

An attenuator (intrinsic terminator) is

located between the promoter and the first gene of the trp cluster

The absence of Trp-tRNA suppress

termination and results in a 103 increase in transcription

An attenuator controls the progression of RNA polymerase into the

trp genes

Attenuation can be

controlled by translation

The leader region of the trp operon has a

14-codon open reading frame that includes two codons for trytophan

The structure of RNA at the attenuator depends on whether this reading frame is

translated

In the presence of Trp-tRNA, the leader is translated to a

leader peptide, and the attenuator is able to form the hairpin that causes termination

The trp leader region cna exist in

alternative base-paired conformations

The alternatives for RNA polymerase at the attenuator depend on

the location of the ribosome

In the absence of Trp-tRNA, the

ribosome stalls at the tryptophan codons and an alternative secondary structure prevents formation of the hairpin, so that transcription continues

In the presence of tryptophan tRNA, ribosomes translate the

leader peptide and are released

Eukaryotic gene expression is usually controlled at the level

of initiation of transcription by opening the chromatin

Gene expression is controlled principally at the

initiation of transcription

Some transcriptions factors may compete with histones for DNA after

passage of a replication fork

Some transcription factors can recognize their targets in

closed chromatin to initiate activation

The genome is divided into domains by

boundary elements (insulators)

Insulators can

block the spreading of chromatin modifications from one domain to another

When replication disrupts chromatin structure, chromatin can

reform or transcription factors can bind and prevent chromatin formation

Activators

determine the frequency of transcription

Repressor

a protein that inhibits expression of a gene

Repressors may act to

prevent transcription by binding to an operator site in DNA or to prevent translation by binding to RNA

Positive control

The default state of genes that are under positive control is that they cannot be expressed unless a positive regulator is bound

The activity of a positive regulatory transcription factor is controlled in

various ways

Activators work by

making protein-protein contacts with the basal factors

Activators may work via

coactivators

Activators are regulated in

many different ways

Antirepressor

a positive regulator that functions in opening chromatin

Architectural protein

a protein that, when bound to DNA, can alter its structure (e.g, introduce a bend)

• may have no other function