Regulation Of Gene Expression

In Prokaryotes
Metabolism
Highly efficient genetic mechanisms turn
genes depending on the cells metabolic
needs
Organisms regulate gene activity in a
variety of cellular responses including
replication, recombination, and DNA repair

Gene Expression
Constitutive genes expressed constantly,
house-keeping genes
Needed under certain environmental
conditions:
inducible
repressible

Inducible Genes in Prokaryotes

Bacteria energy source, utilize any of
several carbohydrates (glucose, sucrose,
galactose, arabinose, lactose)
Glucose is prefferential
No glucose utilize other carbohydrates
Glucose, no lactose no need for
expression
No glucose, lactose - genes expressed

Inducible Genes in Prokaryotes

Inducible:
No lactose genes off
Lactose synthesis of beta-galactosidase

Catabolic (degradation) pathways usually

inducible
Induction alters the rate of enzyme
synthesis, not enzyme activity
Induction occurs on transcriptional level

Repressible Genes in Prokaryotes

Anabolic pathways synthesis

Cell can make tryptophan (5 enzymes)
If no tryptophan active transcription
Tryptophan present genes turned off
Repression

Positive And Negative Control

Of Gene Expression
Regulator genes encode products that
regulate the expression of other genes
Positive control product of regulator gene
turn on expression (activators)
Negative control product of regulatory
gene turn off expression of structural genes
(repressors)

Positive And Negative Control

Of Gene Expression
Inducer binds to activator
Co-repressor binds to repressor
Change in 3-D structure of proteins
Allosteric transition
Conformation changes

The lac operon

A model of negative and positive regulation in E.
coli
Nobel prize for Jacob and Monod
Regulates utilization of lactose sugar
lactose must be present
glucose (preferred sugar) must be absent
repression of lac operon by glucose is example of catabolite
repression (actually, positive control!)

example of negative regulation

Induction
lac operon is an example of an inducible system
Induction
relief of repression by action of an inducer
inducer binds allosterically to repressor, altering its
conformation and binding capacity

During repression there is no transcription of

structural genes Z, Y, and A
After induction, these genes are transcribed

Lactose Metabolism In E. coli

cis acting regulatory site
trans-acting elements binding molecules
that control transcription of the gene cluster
can be positive or negative regulation
The three structural genes and the adjacent
regulatory site constitute the lactose, or lac,
operon

lac Operon

The lac operon components

lac structural genes

Z: -galactosidase
Y: permease
A: transacetylase
transcribed as single,
multigenic mRNA

lac regulatory components

I: encodes lac repressor
P: promoter site for RNA
polymerase binding
O: operator site to which
Lac repressor binds

P, O, Z, Y, and A
components constitute
lac operon, called lacO,
lacZ, etc.

Background Activity of Lactose

Operon
Lactose ---- beta-galactosidase---allolactose
Allolactose inducer, binds to repressor,
release operator

lac Operon
Lederberg - lac operon mutants (deletions, lac-)
lacZ lacY lacAGene order Z-Y-A
All three genes are transcribed as a single unit
polycistronic RNA
Transcription and translation can occur almost
simultaneously!

Genetic analysis
The lac operon was discovered through a combination
of biochemical and genetic analyses
Mutations in repressor and operator cause
misregulation of lac operon.
OC mutations result in constitutive expression because
repressor can not bind
said to be cis-acting (adjacent sequence)

I mutations result in constitutive expression because no

functional repressor is produced
said to be trans-acting (not adjacent)

Genetic analysis

IS mutations (super-repressors) result in

permanent repression because inducer can
not bind to repressor.
P region, promoter
between I and O
region for binding of RNA polymerase

Repressor and CAP-cAMP binding sites

differ in nucleotide sequence
both show rotational twofold symmetry (partial
palindrome may form a hairpin)

Genetic Proof of the Operon

Model
Predictions:
The I gene produces a product
The O region is involved in regulation but does
not make a product
The O region must be adjacent to the structural
genes to regulate transcription

Genetic Proof of the Operon

Model
Created merozygotes (temporary partial
diploid, small segment of circular bacterial
genes) using F plasmid
Found a mutant IS that produces a product
that cannot interact with lactose structural
genes permanently repressed

Figure 16-6

Copyright 2006 Pearson Prentice Hall, Inc.

h merozygotes
te regulatory gene

Need to k
whether
transcript
or not?

Lac Operon Mutations

Wild type I+P+O+Z+Y+A+
Partial diploids (merozygotes) through
Ffactor
F I+P+O+Z+Y+A+
I+P+O+Z-Y-AI+P+O+ Z-Y-AI+P+O+ Z+Y+A+

inducible, Z+Y+A+ dominant

Lac Operon Mutations

I+P+O+Z+Y+A+ - I+/I- - inducible, I+ dominant over II-P+O+Z+Y+A+
I controls expression in cis and trans
Operator mutations: Oc acts only in cis
I+P+OcZ-Y-AI+P+O+Z+Y+A+

inducible

I+P+OcZ+Y+A+
I+P+O+Z-Y-A-

constitutive

-galactosidase
production
Genotype
(a)
(b)
(c)
(d)

I + O+ Z+/ F I - O+ Z+
I - Oc Z +/ FI - O+ ZI s Oc Z +/ FI + O+ Z+
I - O+ Z +/ F I - O+ Z+

No Lactose

On Lactose

Catabolite repression
Catabolite repression is superimposed on the lac
and related operons
glucose is preferred carbon source
induction occurs only in absence of glucose

Two components
Cyclic adenosine-3,5- monophosphate (cAMP)
effector protein
when glucose level is low, cAMP concentration rises because
AMP is not converted to ATP

catabolite activator protein (CAP)

CAP is activated allosterically by cAMP
active CAP binds to CAP site of lac operon, facilitating binding
of RNA polymerase

lac Operon: Positive Control

Lactose -------- glucose + galactose

If lactose and glucose both present?
The cell prefers glucose.
If it is available, lac operon is not activated

lac Operon: Positive Control

Lac promoter P has two binding sites:
For RNA polymerase
For CAP-cAMP complex
CAP-cAMP complex must be present in
binding site for the operon to be induced
normally positive control
Intracellular concentration of cAMP is sensitive
to presence/absence of glucose

lac Operon: Positive Control

In order to bind to the promoter, CAP must
be bound to cAMP (cyclic adenosine
monophosphate)
The level of cAMP dependes on enzyme
adenyl cyclase which catalyzes conversion
of ATP to cAMP
Glucose inhibits the activity of adenyl
cyclase

The Tryptophan Operon:

Repressible Gene System
1953, Monod et al
If tryptophan is present in sufficient
quantities, the enzymes necessary for its
synthesis are not produced

The Tryptophan Operon:

Repressible Gene System
No tryptophan RNA polymerase binds to
promoter synthesis of RNA transcript
Tryptophan present TrpR produces
repressor tryptophan binds to repressor
complex binds to operator O prevent RNA
polymerase from binding no synthesis

trp Operon
Five structural genes trpA, B, C, D, E
In presence of tryptophan, all are repressed,
no enzymes are produced
Repressor is present that is normally
inactive
Repressor binds to tryptophan and attains
new configuration that allow the complex to
bind to the operator, repressing transcription

trp Operon
Since the regulatory complex inhibits
transcription of the operon, the repressible
system is under negative control
As tryptophan participates in repression, it
is a corepressor
End product regulation

trp Operon
trpR+ - encodes a functional repressor
molecule
trpR- - no repression occurs
trpO+
trpO trpO-/F O+ - does not restore repression

trp Operon
Structural genes are transcribed as a polycistronic
message
Promoter trpP binding site for RNA polymerase
trpO operator region binds the repressor
Transcription is initiated within the overlapping
trpP trpO region and proceeds along a leader
sequence 162 bp before the structural genes
Within regulatory sequence, another regulatory
site - attenuator

trp Operon
Even if tryptophan is present, initiation of
transcription still occur initial portion of
mRNA is transcribed (the 5-leader
sequence)
In the presence of high concentration of
tryptophan mRNA synthesis is terminated at
a point ~140 nucleotides
________________________________

trp Operon
This process is called attenuation (reducing
in strength)
Deletions in +115-140 region abolish
attenuation
Initial transcript can fold into two types of
hairpins

trp Operon
The leader sequence has AUG UGG UGG codons
(UGG trp)
Tryptophan present tRNAtrp is also present,
translation proceeds past these codons, terminator
hairpin is produced
No tryptophan no tRNAtrp transcription stalls
Attenuation is overcome and
_________________________________________

If transcription and translation occurs

simultaneously:

Attenuation In Bacillus subtilis

Slightly different mechanism from E. coli
Protein trp RNA-binding attenuation protein
TRAP binds to tryptophan if it is present in the
cell
TRAP consists of 11 subunits each binds one
Trp
When fully saturated, TRAP binds to 5-leader
sequence prevent anti-termination pin to form

Figure 16-14 Copyright 2006 Pearson Prentice Hall, Inc.

Figure 16-14a

Copyright 2006 Pearson Prentice Hall, Inc.

Figure 16-14b

Copyright 2006 Pearson Prentice Hall, Inc.

AP Protein
Another protein AP anti-TRAP provides
a signal that tRNAtrp is uncharged no trp
in cell
Uncharged tRNAtrp induces another operon
to express AT gene
AT associates with TRAP, inhibit binding to
leader RNA sequence

ara Operon
Controlled by a regulator protein that has
both positive and negative control

Figure 16-15

Copyright 2006 Pearson Prentice Hall, Inc.