Week 2A - Regulation of the Bacterial Gene Expression - The Operon Part I Flashcards
Trans-acting factors
generally genes, the product of which can function on any copy of its target DNA
• code for tRNA or rRNA
• ie a protein or RNA molecule that diffuses AWAY from the location of synthesis to act elsewhere
DNA
• a DNA sequence that contains a gene
– this gene codes for a protein (or microRNA or other diffusible molecule) that will be used in the regulation of another target gene.
Cis-acting DNA sequences
a site that affects the activity ONLY of sequences on its OWN molecule or DNA (or RNA)
• usually implies that the site does not code for protein
• ie promoters, operators, and terminators
• DNA sequence that functions solely as a DNA sequence, have only LOCAL effects
Structural gene
a gene that codes for any RNA or protein product other than a regulator
Regulator gene
a gene that codes for a product (typically protein) that controls the expression of other genes (usually at the level of transcription) by binding to particular sites on DNA
(cis-acting sequences)
Operators
the sites on DNA are usually located just upstream of the target gene
Regulation in a positive manner
- turns the gene ON
* an activator
Regulation in a negative manner
- turns the gene OFF
* a repressor
In negative regulation…
a trans-acting repressor protein binds to a cis-acting operator to prevent a gene from being expressed • repressor and operator overlap
In negative regulation,
in the absence of a repressor, a gene is
ON by default
The most common type of regulation in bacteria
negative regulation
with a repressor
In positive regulation…
a trans-acting transcription factor activator
is required to bind at the
cis-acting promoter
in order to enable RNA polymerase to initiate transcription
• operator and activator don’t overlap
In positive regulation,
in the absence of the positive regulator
(transcription factor)
a gene is…
OFF by default
The most common type of regulation in eukaryotes
positive regulation
A gene that encodes an enzyme may be regulated by
the concentration of the substrate or product
A bacteria avoids synthesizing the enzyme of a pathway in the absence of the
substrate (avoid wasting of energy), but is ready to produce the enzymes if the substrate (inducer) should appear
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
4 combinations of regulatory circuits
- negative inducible
- negative repressible
- positive inducible
- positive repressible
Induction
Negative control
negative control = act on repressor active repressor repressed by inducer --> repressor inactivated to get induction
Induction
Positive control
positive control = act on activator inactive activator induced by inducer to be active --> activator activated to get induction
Repression
Negative control
negative control = act on repressor inactive repressor binds corepressor repressor active --> repressor activated to get repression
Repression
Positive control
positive control = act on activator active activator binds corepressor to become inactive --> activator repressed to get repression
Unifying theme: regulatory proteins are
trans-acting factors that recognize
cis-acting elements usually upstream of the gene
Operon
functioning unit of genomic material containing a cluster of genes under the control of a single regulatory signal or promoter
• eg genes for E. coli metabolism
Genes in an operon are transcribed together into a
polycistronic mRNA strand
The result of an operon is that all the genes are
either expressed together
or not at all
Genes coding for proteins that function in the same pathway are often located
adjacent to one another and controlled as a single unit that’s transcribed into a polycistronic mRNA within an operon
lac operon
proteins products of this operon enable bacteria to take up and metabolize β-galactoside sugars
(ie lactose)
• has structural genes (lac Z Y A)
lacZ
encodes the enzyme β-galatctosidase which breaks down β-galactoside
(lactose to glucose and galactose)
lacY
enables β-galactosidase permease
transports β-galactosides into the cell
lacA
encodes β-galactoside transacetylase
transfers acetyl groups from acetyl-coA to β-galactosides
β-galactosidase
breaks down β-galactoside
disaccharide –> 2 monosaccharides
β-galactoside sugars are
• the substrates of the lac operon
• the inducers
(add = genes transcribed
use up = switch off)
Addition of specific β-galactosides
induces transcription of all 3 genes of the lac operon
The lacmRNA is extremely
unstable - restricts the amount of protein made
–> induction can be rapidly reversed
Promoter
binds RNA polymerase
Operator
binds repressor or activator
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
The lac repressor protein is a
tetramer of identical subunits coded by the laci gene
The lac repressor is encoded by the
laci gene
which is an independent transcription unit with its own promoter and terminator
• overlaps with the promoter region where RNA polymerase binds
• laci makes the monomer which forms a tetramer
The laci gene
is an independent transcription unit
with its own promoter and terminator
Constitutive expression
a state in which a gene is expressed continuously
• eg problem with laci gene = no lac repressor = constitutively expressed
In the absence of β-galactosides
the lac operon is expressed only at a very low (basal) level
An inducer functions by
converting the repressor into a form with lower operator affinity
• the repressor is usually bound, so lac usually off until lactose comes along to change the shape of the repressor
Repressor has 2 binding sites
- 1 for the operator DNA
* 1 for the inducer
Repressor is inactivated by an
allosteric interaction in which binding of inducer at its site changes the properties of the DNA-binding site (allosteric control)
• lactose permeates bacterial cell, the binds lac repressor –> changes shape so it has no affinity for the operator
The true inducer is
allolactose, not the actual substrate of β-galactosidase
Gratuitous inducer
inducers that resemble authentic inducers of transcription, but are not substrates for the induced enzymes (eg IPTG)
(here resembles lactose but does the same thing)
Cis-acting constitutive mutations identify the
operator
Mutations in the operator cause
constitutive expression of all 3 lac structural genes
(repressor binds at operator)
• these mutations are cis-acting and affect only those genes on the contiguous stretch of DNA
Mutations in the promoter that prevent expression of lacZYA are
uninducible and cis-acting
Cis-dominant
a site or mutation that affects the properties only of ts own molecule of DNA, often indicating that a site does not code for a specific product
Trans-acting mutations identify the
regulator gene
Mutations in the laci gene are
trans-acting and affect expression of all laczYA clusters in the bacterium
Mutations that eliminate laci function cause constitutive expression and are
recessive
laci-
Mutations in the DNA binding site of the repressor are constitutive because
the repressor cannot bind the operator
Constitutive expression can come from
- mutations in the operator (where the repressor binds)
* mutations in the laci gene
The lac repressor is a
tetramer made of 2 dimers
A single repressor subunit can be divided into
- the N-terminal DNA-binding domain
- a hinge
- the core of the protein
The DNA-binding domain of the lac repressor contains
2 short α-helical regions that bind the major groove of DNA
helix-turn-helix motif
The core of the lac repressor contains
- the inducer-binding site
* the regions responsible for multimerization
In the lac repressor, monomers form a dimer by making contacts between
core subdomains 1 and 2
Dimers form a tetramer by interactions between
the tetramerization helices
The lac repressor is
large compared to DNA
lac repressor binding to the operator is regulated by
an allosteric change in conformation
lac repressor binding to the operator
• lac repressor protein binds to the double-stranded DNA sequence of the OPERATOR
(operator is palindromic 26bp)
• each inverted repeat of the operator binds to the DNA-binding site of one repressor subunit
• symmetry of the operator matches the symmetry of the repressor
• inducer binding causes a change in repressor conformation that reduces its affinity for DNA and releases it from the operator
(probably from changing the hinge helices)
The operator is
a palindromic sequence of 26 bp
• dyad symmetry
Each inverted repeat of the operator binds to
the DNA-binding site of one repressor subunit
Symmetry of the operator matches
the symmetry of the repressor
Inducer binding causes a change in repressor conformation that
reduces its affinity for DNA and releases it from the operator
• probably by changing the hinge helices
A dimer of the lac repressor binds
2 halves of DNA
• its a tetramer because there’s more than 1 operator to bind to
lac operators
the original operator (lacO1)
auxiliary operators (weaker)
• 410 bp downstream (lacO2)
• 88 bp upstream (lacO3)
because there’s 3 operators = repressor tetramer binds to more than 1
The lac repressor binds to
3 operators and
interacts with RNA polymerase
Each dimer in a repressor tetramer can
bind an operator
• so the tetramer can bind 2 operators simultaneously
Full repression requires
the repressor to bind to an additional operator
downstream (lacO2) and
upstream (lacO3) as well as
to the primary operator at the lacZ promoter
(bends to bind both the original and lacO3 = locks in transcriptional repression)
Binding of the repressor at the operator
actually stimulates binding of RNA polymerase at the promoter but precludes transcription
The operator competes with
low-affinity sites to bind repressor
The large number of low-affinity sites ensures
that all repressor protein is bound to DNA
at random sites
Repressor binds to the operator by
moving from a low-affinity site rather than by equilibrating from solution
(previously bound to DNA at random site, moving to operator after its already bound to DNA is easier than coming from solution)
In the absence of the inducer
the operator has an affinity for repressor that is 10^7 times that of a low-affinity site
The level of 10 repressor tetramers per cell ensures that
the operator is bound by repressor 96% of the time
Induction reduces…
the affinity for the operator to 10^4 times that of low-affinity sites, so that the operator is bound only 3% of the time
Transcription is regulated by the interaction between
trans-acting factors (product of a gene) and
cis-acting sites (DNA sequence)
Bacterial genes coding for proteins whose functions are related may be organized in a cluster that is transcribed into a
polycistronic mRNA
from a single promoter
• control of this promoter controls all the genes
The unit of regulation is called
an operon
• contains structural genes and cis-acting elements
Initiation of transcription is regulated by interactions that
occur in the vicinity of the promoter (at the operator)
The ability of RNA polymerase to initiate transcription is prevented
by a repressor
The ability of RNA polymerase initiate transcription is activated
by an activator
Genes that are active only when the regulator is bound to them are said to be
under positive control
Genes that are off when the regulator is bound are said to be
under negative control
The ability of a repressor to bind its operator sequence is often regulated by
small molecules which provide a second level of gene regulation
If the repressor regulates genes that encode for enzymes, the system may be
induced by the enzyme substrates
(negative inducible - prevents repressor from binding)
–> genes switch ON
If the product of the corepressor enables the regulator to bind the operator
= negatively repressible
–> gene turned off
Binding of the inducer or corepressor to its site on the regulatory protein produces
an allosteric change in the structure of the DNA-binding domain of the regulatory protein
The lactose pathway in E. coli operates by negative induction
• when an inducer, the substrate β-galactoside diminishes the ability of repressor to bind its operator, transcription and translation of the lacZ gene then produce β-galactosidase, the enzyme that metabolizes β-galactosides
