Lecture 14, Tues, Feb 24

Regulation of gene expression

! Key points from Lecture 13 (mutations)
! Briefly: promoters & transcription
! Prokaryote regulation
! inducible gene regulation: lac operon
! lac repressor
! CAP activator

! repressible gene regulation: trp operon

! Eukaryotic gene expression

! promoters and enhancers

! RNA interference
! newly recognized method to regulate gene
expression
! also useful genetic tool (medical tool?)

Klug
Chapter 15

Class business
Midterm grades uploaded to Ted
Reflects +7 added to grade that is written on paper
exam
Q6 regrade: for branched diagram, it is required to
show how you multiplied together the individual
phenotypes (or we also accepted genotypes)

Midterms available from Bonner 4th floor

Outside Bonner 4334 if waiver
Inside Bonner 4334 if no waiver

Professor Troemel office hours today

10-11am in Bonner 4202

Key points from Lecture 13

Mutant lecture: Ch. 14 in Klug
! Mutations: chain of effects from DNA protein - function - dominant/recessive
! Causes of mutations
! internal/spontaneous
! external/induced

! DNA repair pathways

Chain of events from mutation to outcome
Genetic code will be provided on a test

Clicker Q1: What kind of mutation

is this?
TAT AAA CAT GAC to
TAA AAA CAT GAC
A) substitution, missense
B) substitution, silent
C) substitution, nonsense
D) Insertion
E) Frameshift

What kind of mutation is this?

! TAT AAA CAT GAC to
! TAT GAA ACA TGA C
! missense
! nonsense
! silent
! deletion
! insertion
! frameshift
What other problem does this frameshift cause?
premature stop

Asp to stop

What kind of effect can this mutation

have on protein function?
! Asp to stop
! gain of function?
! loss of function?

! What kind of loss of

function might this be?
! what if stop happens at
beginning of the protein?
! null (complete lof)
Will this mutation likely be dominant or recessive?

Lecture 14, Tues, Feb 24

Regulation of gene expression
! Key points from Lecture 13 (mutations)
! Prokaryote regulation
! inducible gene regulation: lac operon
! lac repressor
! CAP activator

! repressible gene regulation: trp operon

! Eukaryotic gene expression

! promoters and enhancers

! RNA interference
! newly recognized method to regulate gene
expression
! also useful genetic tool (medical tool?)

Klug
Chapter 15

How is gene expression regulated?

! Until now: gene transmission, interactions
mapping, mutations and consequences
! But how are those genes regulated?
! Only certain genes are expressed at certain times
in certain places in certain amounts
! e.g. regulation of amount
! some E. coli proteins present at 5-10 molecules per cell,
while others are 100,000 copies per cell

few protein copies

many protein copies

Cell-type specific gene expression

! Only certain genes are expressed at certain times in
certain places in certain amounts
! e.g. cell-type specific expression: Remember in multicellular organisms, DNA for all genes is in all cells,
but only EXPRESSED in certain cells

pancreatic cell makes insulin

retinal cell doesnt

(even though it has the gene)

pancreatic cell doesnt make pigment

retinal cell makes retinal pigment

How do organisms respond to

changing environmental conditions?
! One mechanism of response is to regulate
expression of mRNA from genomic DNA
regulate this step

probably predominant level of

regulation in prokaryotes

Figure 1-8

Transcription/promoters

Repressor
proteins

Figure 12-8

Negative regulation of transcription

What makes us different from

chimps?
Humans and chimps share ~98% of
genes
the major difference between us and
chimps is likely to be gene regulation

Regulation of gene
expression

ON
OFF

flipping a switch
! Bacteria (and other microorganisms) adapt to
changing environmental conditions
! Inducible system
! If certain sugars are present in the environment (e.g.
lactose), bacteria express enzymes to utilize those sugars
! only make enzymes when substrates are present
! Inducible enzymes (induced by substrate)

! Repressible system (end product)

! If tryptophan is present in the environment, it will repress
enzymes that synthesize tryptophan
! only make enzymes when end-product is absent
! repressible enzymes (repressed by end-product)

Can also have a combination of these systems

Negative vs. positive control of

transcription
Negative regulation of transcription
Lactose inhibits
the repressor:
Inducible system

Repressor
protein

Lactose utilization genes

Example of inducible gene

regulation in bacteria: lac operon
Genes are found in operons
lac operon allows bacteria to utilize lactose
several genes contained in this operon
Figure 15-1

Lactose is processed into

galactose and glucose by lacZ
gene
Figure 15-2

lacZ encodes betagalactosidase enzyme,

which cleaves lactose into
galactose and glucose

lacZ (and lacY and lacA) only

made when lactose is available

Constitutively OFF mutants

! Normally lac operon induced in the presence
of lactose
! lacZ, lacY, lacA transcribed together
! therefore, regulated together

! Mutants isolated that cannot use lactose as

carbon source

Figure 15-1

lacZ- mutant

lacY- mutant

OFF

ON

Constitutive ON lac mutants:

lacI! Key insights from regulatory mutants:
! constitutive mutant: operon is always ON
! lac enzymes expressed in absence of lactose

X
Figure 15-1

lacI is a repressor
Constitutive mutant: lacI- acts in trans

Constitutive ON lac mutants:

lacOC
! Key insights from regulatory mutants:
! constitutive mutant: operon is always ON
! lac enzymes expressed in absence of lactose

X
Figure 15-1

Constitutive mutant: lacOC

lacO is the operator

acts in cis

ON

cis vs. trans effects

! cis-acting element
! DNA sequence that regulates expression of a
gene located nearby (on the same chromosome)
! e.g. a promoter, operator

! trans-acting element
! factors (usually proteins) that control gene
expression through a cis element
! e.g. transcription factor activator, repressor

What controls these cis and trans acting elements?

Environmental factors: lactose

Lactose regulates the lac

operon via the lacI repressor
Lactose inhibits the repressor
leads to induction of lac operon

Figure 15-5

Lactose regulates the lac

operon via the lacI repressor

Figure 15-5

Normal regulation
no lactose:

OFF

Lactose regulates the lac

operon via the lacI repressor

Figure 15-5

ON

Normal regulation

Lactose flips the

switch to ON

Allosteric regulation
Lactose causes a conformational
change in lacI repressor so that it can
no longer bind to the operator

Gray blob (lac I repressor)

undergoes conformational
change

lac operon constitutively ON

when repressor or operator is
defective

Figure 15-6

Defective repressor

ON

lac operon constitutively ON in

absence of repressor or operator

Figure 15-6

Defective operator

ON

Phenotypes of lac mutants

Testing the model!

no enzyme Zno repressor Ioperator

constitutively
on

OC

Model makes predictions - can test this with merozygote

Diploid for certain genes: can ask about dominant/recessive
What would happen if you combined I- and I+?

Clicker Q2: What would happen if you

combined I- and I+?

A:
B:

+
+

+
-

C:

D:

Phenotypes of lac mutants

cis/trans elements

no enzyme Zno repressor Ioperator

constitutively
on

OC

What would happen if you combined I- and I+?

B) Rescues - looks like wild-type, I+ is dominant
What would happen if you combined OC and O+?

Clicker Q3: What would happen if you

combined O+ and OC?

A:

?
+

B:

+
-

C:

D:

Phenotypes of lac mutants

cis/trans elements

no enzyme Zno repressor Ioperator

constitutively
on

OC

What would happen if you combined I- and I+?

B) Rescues - looks like wild-type
What would happen if you combined OC and O+?
A) Constitutively ON, like OC alone, OC is dominant

Phenotypes of lac mutants

no enzyme Zno repressor Ioperator

constitutively
on

OC

Review other predictions

in your textbook

Video of lac operon

http://www.youtube.com/watch?
v=iPQZXMKZEfw
http://www.youtube.com/watch?
v=W6s7I3I0Kh4&feature=related

cis vs. trans effects

cis-acting element
DNA sequence that regulates expression of a
gene located nearby (on the same chromosome)
e.g. a promoter, operator

trans-acting element
factors (usually proteins) that control gene
expression through a cis element
e.g. transcription factor activator, repressor

Clicker Q4: A mutation in the P

region of the lac operon would
most likely result in
A) constitutive expression of lac, as the
repressor cannot bind.
B) constitutive expression of lac, as
lactose cannot bind.
C) reduced expression of lac.
D) production of nonfunctional galactosidase.
E) no change in the expression of lac.

Clicker Q4: A mutation in the P region

of the lac operon would most
likely result in
Answer:
C. reduced expression of lac.
Explanation:
The promoter (P) allows RNA polymerase to
bind and begin transcription of lac. A change
in P would be likely to affect RNA polymerase
binding and reduce lac transcription.

What do E. coli do if they have

BOTH lactose and glucose?
Lactose is converted to
galactose and glucose
Galactose is converted
to glucose
Glucose is a preferred
carbon source
How is this controlled?
Figure 15-2

Another regulator of the lac

operon: CAP
Catabolite activating protein (CAP)
activates expression of lac operon
binds to promoter

CAP binds promoter better with cAMP

cAMP levels are lower in presence of glucose
glucose inhibits adenylate cyclase, which makes
cAMP

Another regulator of the lac

operon: CAP

Figure 15-8

Another regulator of the lac

operon: CAP

Figure 15-8

Combination of positive and negative regulation determines

lac operon transcription

Clicker Q5: The lac operon is

normally expressed:
A) in the absence of both lactose and glucose.
B) in the absence of lactose and the presence
of glucose.
C) in the presence of lactose and the absence
of glucose.
D) in the presence of both lactose and glucose.
E) None of the above.

Clicker Q5: The lac operon is

normally expressed:
Answer:
C. in the presence of lactose and the
absence of glucose.
Explanation:
For lac expression, the presence of lactose is
necessary to inactivate the repressor. The
absence of glucose allows CAP to form a
complex with cAMP, bind to the CAP site,
and facilitate RNA polymerase binding and
lac transcription.

54th anniversary of the lac operon

! Jacob & Monod elucidated this regulation
! Celebrated 3 years ago in the journal Science
where Jacob reflected on their findings:

! Thus did we discover a
mechanism

fundamental to all living beings from their
very beginnings, and that would persist as
long as they exist... More than ever, research
seemed to be identified with human nature!
It was by far the best means found by man to
face the chaos of the universe.
Success of
night science
modern lab technique: blue/white selection

Gene regulation
can have repressors and activators
sometimes several of each

Combinatorial control

Lecture 14, Tues, Feb 24

Regulation of gene expression
! Key points from Lecture 13 (mutations)
! Prokaryote regulation
! inducible gene regulation: lac operon
! lac repressor
! CAP activator

! repressible gene regulation: trp operon

! Eukaryotic gene expression

! promoters and enhancers

! RNA interference
! newly recognized method to regulate gene
expression
! also useful genetic tool (medical tool?)

Klug
Chapter 15

Example of repressible gene

regulation in bacteria: trp operon
Energetically favorable to repress gene
expression for tryptophan (trp) biosynthesis if
trp is available from media

Figure 15-9

Example of repressible gene

regulation in bacteria: trp operon
Energetically favorable to repress gene
expression for tryptophan (trp) biosynthesis if
trp is available from media

Figure 15-9

ON

Example of repressible gene

regulation in bacteria: trp operon
! Absence of tryptophan

Figure 15-9

Example of repressible gene

regulation in bacteria: trp operon
! Presence of tryptophan

Figure 15-9

OFF

Lecture 14, Tues, Feb 24

Regulation of gene expression
! Key points from Lecture 13 (mutations)
! Prokaryote regulation
! inducible gene regulation: lac operon
! lac repressor
! CAP activator

! repressible gene regulation: trp operon

! Eukaryotic gene expression

! promoters and enhancers

! RNA interference
! newly recognized method to regulate gene
expression
! also useful genetic tool (medical tool?)

Klug
Chapter 15

Gene regulation is more

complicated in eukaryotes
! Gene expression has to be regulated in
amount, time AND place

pancreatic cell makes insulin

retinal cell doesnt

(even though it has the gene)

Eukaryote vs. Prokaryote

e.g. Animal cell

Figure 2-1

* more cell organization: e.g. nucleus,

mitochondria, ER, Golgi
* Usually diploid and several
linear chromosomes
* DNA packaged with histones

Bacterial cell

Figure 2-2

* Haploid and one circular

chromosome

BOTH have doublestranded DNA!

Gene regulation can

occur at many stages
in eukaryotes
Rule in biology:
if it can be regulated, it will be!

Figure 15-11

Transcription in eukaryotes
can be regulated by cis and
trans elements

Eukaryotic gene regulation at

level of cis-acting elements
! Promoters are adjacent to genes
! specify basal expression of genes

! Enhancers are farther away

Can alter transcription of genes

Regulation at level of trans-acting

elements
! Trans-acting elements bind to the cisacting elements

Human metallothionein IIA gene promoter

Figure 15-16

Combinatorial control

Regulation of mRNA splicing

Due to alternative splicing,

can make 2 different
hormones with different
structures, locations, and
functions!

Figure 15-19

Can also have regulation

of mRNA transport,
stability, translation into
protein, etc, etc

Figure 15-11

Lecture 14, Tues, Feb 24

Regulation of gene expression
! Key points from Lecture 13 (mutations)
! Prokaryote regulation
! inducible gene regulation: lac operon
! lac repressor
! CAP activator

! repressible gene regulation: trp operon

! Eukaryotic gene expression

! promoters and enhancers

! RNA interference
! newly recognized method to regulate gene
expression
! also useful genetic tool (medical tool?)

Klug
Chapter 15

RNA interference: a newly

recognized mode of gene regulation
! Shaking up the central dogma

dsRNA
~21-24 nucleotides

How was RNAi discovered?

(and why was it missed for so long?)
! Fire and Mello injected unc-22 RNA into C.
dsRNA
elegans

! Expected antisense RNA to block function

and sense to not block function
! instead, both blocked function, and dsRNA
blocked it best of all! Nature 1998 paper
Nobel prize in medicine, 2006

Mechanisms of RNAi
Still being worked out - models likely to
change
Important to know what RNAi IS and
how it is useful
perhaps exists for defense against viruses
useful for genetic studies in lab
also may be a therapeutic in the clinic
several companies focusing on this

Gene regulation by RNAinduced gene silencing

Figure 15-21

Lecture 14, Tues, Feb 24

Regulation of gene expression
! Key points from Lecture 13 (mutations)
! Prokaryote regulation
! inducible gene regulation: lac operon
! lac repressor
! CAP activator

! repressible gene regulation: trp operon

! Eukaryotic gene expression

! promoters and enhancers

! RNA interference
! newly recognized method to regulate gene
expression
! also useful genetic tool (medical tool?)

Klug
Chapter 15