BIOSC20187

Part II: Molecular Genetics

• Lectures by Erin Green, Ph.D.
• *New third time added* Office Hours
in BSLC 216: Tues 11am-12pm,
Thursday 3-4pm; Friday 1:15-2:15pm
• eringreen@uchicago.edu
 Part II, Lecture 6:

Regulation of gene
expression in bacteria
 Part II, Lecture 6: Learning objectives
• Describe how bacterial genes are organized into operons and co-transcribed as
polycistronic mRNA

• Understand how elements in the 5’ UTR upstream of bacterial ORFs influence gene
transcription

• Understand the difference between promoter and operator sequences

• Describe how allosteric changes in the structure of DNA binding proteins can
influence gene transcription outcomes

• Describe the function and control of the lac operon through both negative and
positive regulation

• Understand how mutations in cis and trans acting components of the lac operon
influence gene regulation
Two bacterial strains with the same genotype
can have very different phenotypes under
different environmental conditions
Two bacterial strains with the same genotype
can have very different phenotypes under
different environmental conditions

This phenotypic heterogeneity can be largely

attributed to changes in gene expression
under different environmental conditions!
The central dogma explains the flow of genetic
information from DNA to mRNA to protein
The central dogma as it applies to bacteria
 Only one strand of DNA is the template for gene
transcription, but the strand can vary with the gene

The resulting RNA sequence is:

• complementary to the
template strand
• identical (except for the use
of uracil in place of thymine)
to the non-template strand

Template strand = non-coding strand

Non-template strand = coding strand
Transcription begins following binding of RNA
polymerase holoenzyme to the promoter
Transcription terminates when RNA polymerase senses
certain intrinsic features in the nucleotide sequence
Bacteria couple transcription and translation
Allows the cell to make lots of protein very quickly!
 Bacteria organize their genes into
polycistronic operons

Typically, operons encode a group of

genes whose products are needed at
the same time.
Examples include:
• Metabolic pathways (synthesis or
degradation)
• Biosynthesis of structural components
of the cell
• Virulence factors (pathogens)
 Why do bacteria regulate gene expression?
Bacteria are opportunists!

• Making mRNA and proteins when you don’t need them is

energetically expensive
• By sensing and responding to changes in the environment,
bacteria can adapt to complex environmental conditions
(nutrition, stress, etc.)
• Encoding regulatory systems in operons allows bacteria to quickly
turn on or off sets of functionally related genes only when
necessary
All operons have promoters – site of RNA pol binding
 Interactions between RNA Polymerase and
promoter sequences are sometimes stabilized
by activator proteins
Some 5’ UTRs have operator sequences
 Repressor proteins bind operator sequences and
inhibit RNA polymerase from initiating transcription
 Two essential requirements are necessary
for any regulatory system in bacteria
1. They must be able to toggle on or off, like a switch, the
transcription of each specific gene or group of genes.

2. They must be able to recognize environmental

conditions in which they should activate or repress the
transcription of the relevant genes.
Both activator and repressor proteins must be
able to recognize when environmental
conditions are appropriate for their actions
 Activators and repressors must be able to
exist in two states: one that can bind its DNA
targets and another that cannot
Effectors can have Inducer activates
different impacts activator

1. May activate binding of

activator (inducer)
2. May inactivate binding
of repressor (inducer) Transcription
3. May activate binding of Co-repressor
activates repressor
repressor (co-repressor)
4. May inactivate binding
of activator (inhibitor)
X
No Transcription
Early work characterizing the lac operon
of E. coli established a model for
understanding bacterial gene regulation
François Jacob and Jacques Monod
The lac operon encodes genes required for
the utilization of lactose

LacI = repressor of operon

P = promoter (site of RNA polymerase binding)
Operator = site of LacI bidning
LacZ = beta-galactosidase (breaks down lactose into glucose and
galactose)
LacY = permease (pumps lactose into cell)
LacA = transfers an acetyl group from coenzyme A to a galactoside
molecule, (helps to clean up toxic byproducts of lactose
metabolism)
The lac operon is induced when lactose is present
and controlled by negative regulation
When no lactose is present, LacI binds the
operator sequence upstream of lacZ

RNA polymerase is blocked!

When lactose is present, it binds and
inactivates LacI

RNA polymerase can bind

Operon genes are expressed!
An inactivating mutation in the lac
operator (lacO) would cause:
 Inactivation of the lac operator will prevent the
repressor from binding, cause constitutive expression
Would introducing a second copy of just lacO on a
plasmid reverse phenotype of a lacO- strian?
Operators are cis-acting elements
An inactivating mutation in lacI would cause:
Inactivation of the lac repressor (lacI) will prevent the
repressor from binding, cause constitutive expression
Would introducing a second copy of just lacI
reverse the phenotype of a lacI- strain?
Repressors are trans-acting

The operon is not expressed

Phenotype is reversed
What if an inducer-blind lacI allele (lacIS) were introduced into
a WT strain (when lactose is around). Is the operon expressed?
What if an inducer-blind lacI allele (lacIS) were introduced into
a WT strain (when lactose is around). Is the operon expressed?
lacIS allele is dominant (a super-repressor)!

The operon is not expressed

Bacteria experience diauxic growth under different
conditions – prioritize some nutrients over others
 The lac operon is also controlled by
glucose-dependent positive regulation

1. A breakdown product of glucose

blocks the synthesis of cyclic
adenosine monophosphate (cAMP).
When glucose is high, cAMP levels
are therefore low.

2. Conversely, when glucose levels are

low, cAMP levels are high
 The lac operon is also controlled by
glucose-dependent positive regulation
1. cAMP binds to the regulatory protein
CAP (catabolite activator protein)

2. cAMP-bound CAP binds to the lac

operon promoter

3. DNA-bound CAP is then able to

interact physically with RNA
polymerase and increases that
enzyme’s affinity for the lac
promoter
 In summary, let’s review different lac
operon conditions
• When glucose is present and lactose is low, is the lac operon
transcribed?
 In summary, let’s review different lac
operon conditions
• When glucose is present and lactose is low, is the lac operon
transcribed?
NO
 In summary, let’s review different lac
operon conditions
• When glucose and lactose are both present is the lac operon
transcribed?
 In summary, let’s review different lac
operon conditions
• When glucose and lactose are both present is the lac operon
transcribed?
AT LOW LEVELS
 In summary, let’s review different lac
operon conditions
• When glucose is low and lactose is present, is the lac operon
transcribed?
 In summary, let’s review different lac
operon conditions
• When glucose is low and lactose is present, is the lac operon
transcribed?
YES
The same principles of gene regulation initially
discovered in lac can be applied to many other
bacterial systems
Iron availability Oxygen Antibiotic exposure
(respiration)
 Practice Question
Under conditions when Zinc is limiting, E. coli produces
mRNA encoding the Zinc uptake system ZnuABC, however
no znuABC mRNA is produced under high Zn conditions

1. How do you hypothesize the znuA, B,

and C genes are organized?

2. What is one mechanism that would

explain activation of these genes
during low Zn and repression during
high Zn?
znuABC are encoded in an operon under the
control of the Zn-binding repressor Zur