Chapter 9
Over 200 different cell types
all (almost) have the same have same genome
all doing different things
e.g., Hb genes expressed only in RBC precursors
Differences are due to gene regulation
which genes will be transcribed?
how transcript will be processed?
when mRNA is translated?, how often?
Chapter 9
Molecular Mechanisms of Gene Regulation
9.1 Transcription regulation in Prokaryotes
9.2 Polycistronic genes - lac operon
9.3 Transcriptional termination (attenuation)
9.4 Eukaryotic transcriptional regulation
9.5 Chemical modifications of the DNA
9.6 Regulation via RNA processing and decay
9.7 regulation via translation
9.8 programmed DNA rearrangements in development
© 2006 Jones and Bartlett Publishers
Regulatory sequences in the DNA
Regulatory molecules made by the cell
Other Regulatory molecules
(may come from cell’s environment)
Transcriptional control
on:
off:
actively transcribed
very low background levels
In bacteria (and bacteriophages)
coordinated regulation
synthesis of polycistronic mRNA
would this occur in eukaryotes?
negative control
positive control
default is “on”
default is “off”
Negative control
a regulatory molecule binds to a gene
preventing transcription
repressor
binds to
operator
Negative control
repressor
binds to
operator
region of the gene that binds to the repressor
(often composed of inverted repeats)
Fig 9.1A
Default state of transcription is “on”
Repressor binds to operator and turns gene “off”
© 2006 Jones and Bartlett Publishers
Positive control
a regulatory molecule binds to a gene
facilitating transcription
activator
binds to
activator binding site
Positive control
activator
binds to
activator binding site
The region of the gene that
binds to the activator
Fig. 9.2. In positive regulation, the default state of transcription is "off."
© 2006 Jones and Bartlett Publishers
Induction
a signal from the cell’s environment
results in transcription (turns gene “on”)
inducer
molecule from the cell’s environment
that increases transcription
Repression
a signal from the cell’s environment
inhibits transcription (turns gene “off”)
corepressor
molecule from the cell’s environment
that decreases transcription
Both negative and
positive control
can involve
induction or
repression
negative control-induction
inducer inactivates the repressor
lactose
© 2006 Jones and Bartlett Publishers
often found in
catabolic pathways
Fig 9.1A and B
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negative control-repression
corepressor activates
the repressor
(tryptophan)
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often found in
anabolic pathways
Fig 9.1C
© 2006 Jones and Bartlett Publishers
positive control-induction
inducer activates the activator
© 2006 Jones and Bartlett Publishers
positive control-repression
corepressor inactivates
the activator
© 2006 Jones and Bartlett Publishers
Constitutive expression
The gene is always transcribed
(always “on”)
© 2006 Jones and Bartlett Publishers
Chapter 9
Molecular Mechanisms of Gene Regulation
9.1 Transcription regulation in Prokaryotes
9.2 Polycistronic genes - lac operon
9.3 Transcriptional termination (attenuation)
9.4 Eukaryotic transcriptional regulation
9.5 Chemical modifications of the DNA
9.6 Regulation via RNA processing and decay
9.7 regulation via translation
9.8 programmed DNA rearrangements in development
© 2006 Jones and Bartlett Publishers
metabolism of lactose in E. coli
controlled by the lactose operon
negative control by induction
repsonds to lactose
positive control by induction
responds to glucose
metabolism of lactose in E. coli
controlled by the lactose operon
operon
group of linked genes sharing promoter
and regulatory sequences
transcribed as a polycistronic mRNA
prokaryotic
metabolism of lactose in E. coli
two proteins are required
lactose permease
lacY
get lactose
into the cell
beta(-galactosidase
lacZ
enzyme that cleaves lactose
into galactose and glucose
Fig. 9.3. The "on-off" nature of the lac system
© 2006 Jones and Bartlett Publishers
no lactose - no permease or -galactosidase
add lactose - quick, transient expression of mRNA
both enzymes appear together
inducible transcription of mRNA
(its not on but we can turn it on)
lactose is the inducer
(lactose is what turns it on)
Fig. 9.4A,B. The lac Operon model
© 2006 Jones and Bartlett Publishers
Fig. 9.4C. The lac Operon model
© 2006 Jones and Bartlett Publishers
Fig. 9.4C. The lac Operon model
© 2006 Jones and Bartlett Publishers
lac system summary (pp 322-324)
1.
two kinds of components:
structural
regulatory
i, o, p, z, y, a
regulatory elements
repressor i
operator o
promoter p
structural genes encode for proteins
z
-galactosidase
y
permease
lac system summary (pp 322-324)
2.
Products are coded by polycistronic mRNA.
Linked structural genes, regulatory promoter
and operator make up the lac operon
(we won’t worry with lacA product now)
3.
Promoter mutations (lacP-) eliminate the ability
to synthesize lac mRNA
lac system summary (pp 322-324)
4.
Product of the lacI gene is a repressor which
binds to the operator DNA sequence
5.
When the repressor is bound to the operator,
initiation of transcription of the lac mRNA is
prevented
6.
When the inducer is present, it inactivates the
repressor, permitting RNA polymerase to bind to
the promoter/operator and initiate transcription
of the lac mRNA
lac system summary (pp 322-324)
inducible
can be turned on with lactose
negative
regulation
transcription occurs until turned off
by repressor
positive
regulation
in a few minutes
lac operon
additional observations
• operator has to be very near promoter
(binding of repressor to operator blocks promoter)
• repressor does not need to be near operator
product is a protein that diffuses
• repressor binding > 1000 x repressor-inducer
• b-gal > permease > transacetylase
1.0
0.5
0.2
lac operon
no lac mRNA is made in the presence of glucose
implies that glucose has an
inhibitory effect on lac operon
glucose affects cAMP levels
cAMP made by adenylate cyclase
cAMP binds to cAMP receptor protein (CRP)
mutation in crp gene
or adenylate cyclase
prevents transcription
of lac mRNA
both are needed for lac mRNA
lac operon
cAMP-CRP complex
must be present for induction of lac operon
even with
lacI- mutants
or
lacOc mutants
cAMP-CRP binds to promoter region
Fig. 9.5. Structure of cyclic AMP
© 2006 Jones and Bartlett Publishers
Fig. 9.6. Four
regulatory states of
the lac operon
© 2006 Jones and Bartlett Publishers
lac operon
cAMP-CRP complex
must be present for induction of lac operon
RNA polymerase only binds strongly to promoter
with the cAMP-CRP complex present
Two ways to turn off:
repressor binding to operator
skip next three slides…
..and wait for them to come out as a movie
repressor CRP-cAMP
binding site binding site
lacI
RNA polymerase
binding site
lacP
repressor
binding site
lacO
lacZ
Fig. 9.7. Base sequence of the control region of the lac operon (left)
© 2006 Jones and Bartlett Publishers
R
CRP
repressor
2 binding sites
RNA
Polymerase
R
http://www.ks.uiuc.edu/images/ofmonth/2005-05/lacI-highlight.jpg
lac operon
repression loop
repressor protein
lac operon
cAMP-CRP complex
must be present for induction of lac operon
RNA polymerase only binds strongly to promoter
with the cAMP-CRP complex present
Two ways to turn off:
repressor binding to operator
or
absence of cAMP-CRP complex
lac system
inducible
can be turned on with lactose
negative
regulation
transcription occurs until turned off
by repressor
positive
regulation
only turned on in presence of
CRP-cAMP complex (+)
repressor
binding site
CRP-cAMP
binding site
positive regulator
no RNA polymerase
binding without it
RNA polymerase
binding site
repressor
binding site
glucose present ?
no need to use lactose
Fig. 9.7. Base sequence of the control region of the lac operon
© 2006 Jones and Bartlett Publishers
Table 9.2. Concentration of cyclic AMP
in cells growing in media with the
indicated carbon sources
Low glucose
High cAMP
high CRP-cAMP complex
© 2006 Jones and Bartlett Publishers
lac system
inducible
can be turned on with lactose
negative
regulation
transcription occurs, until turned off
by repressor (normal)
positive
regulation
only turned on in presence of
CRP-cAMP complex (+)
catabolic pathway
lac system summary
regulatory elements
repressor I
lacI
makes repressor which can bind to operator
or to lactose (but not both together)
operator o
lacO
site for repressor binding OR
site for RNA polymerase binding
promoter p
lacP
site for RNA polymerase binding
site for CRP binding
Lac Operon:
the Movie
http://vcell.ndsu.nodak.edu/animations/lacOperon/movie.htm
Regulation of Transcription
negative control
a regulatory molecule binds to the DNA
and prevents transcription
repressor
binds to
operator
Regulation of Transcription
positive control
a regulatory molecule binds to the DNA
and facilitates transcription
activator
binds to
activator binding site
Regulation of Transcription
induction
a signal from the cell’s environment that
results in transcription
inducer
Regulation of Transcription
repression
a signal from the cell’s environment that
inhibits transcription
corepressor
Chapter 9
Molecular Mechanisms of Gene Regulation
9.1 Transcription regulation in Prokaryotes
9.2 Polycistronic genes - lac operon
9.3 Transcriptional termination (attenuation)
9.4 Eukaryotic transcriptional regulation
9.5 Chemical modifications of the DNA
9.6 Regulation via RNA processing and decay
9.7 regulation via translation
9.8 programmed DNA rearrangements in development
© 2006 Jones and Bartlett Publishers
trp operon
anabolic pathway
genes for the making the amino acid tryptophan
negatively regulated by repressor
(like lac operon)
repressed when tryptophan is present
(if you have it, you don’t need to make it)
tryptophan is a co-repressor
(not an inducer like lactose)
operon is repressible
(instead of inducible)
trp operon
structural genes for enzymes
needed for tryptophan biosynthesis
trpE, trpD, trpC, trpB, trpA
regulatory elements
trp o, trp p, trp a, trp L, trp R
Fig. 9.9. The trp operon in E. coli
© 2006 Jones and Bartlett Publishers
Fig. 9.10.
Regulation of the
E. coli trp operon
lots of tryptophan…
….don’t need to make more
© 2006 Jones and Bartlett Publishers
trp operon
While trp genes are being transcribed…
…it starts being translated
early in this transcript are two tryptophan codons
in high [tryptophan]
high [tRNA-trp]
ribosome continues…
transcription is terminated
Fig. 9.13. Mechanism of attenuation in the E. coli trp operon
© 2006 Jones and Bartlett Publishers
trp operon
While trp genes are being transcribed…
…it starts being translated
early in this transcript are two tryptophan codons
in high [tryptophan]
high [tRNA-trp]
ribosome continues…
transcription is terminated
in low [tryptophan]
low [tRNA-trp]
ribosome pauses…
…transcription continues
Fig. 9.13. Mechanism of attenuation in the E. coli trp operon
© 2006 Jones and Bartlett Publishers
Fig. 9.11. Terminal region of the trp attenuator sequence
© 2006 Jones and Bartlett Publishers
trp operon
attenuation
fine-tuning of control of transcription
superimposed on the negative control
Many operons for anabolic pathways
are regulated by attenuators
Would this work in eukaryotes?
Riboswitches
different configurations of the leader mRNA
antiterminator
vs.
terminator
off
on
antiterminator
terminator
Fig. 9.14B. Riboswitch
[After B.A.M. McDaniel, F. J. Grundy, I. Artsimovitch, and T. M. Henkin. 2003. Proc. Natl. Acad.
Sci. USA 100: 3083. © 2003 National Academy of Sciences, U.S.A.]
© 2006 Jones and Bartlett Publishers
Chapter 9
Molecular Mechanisms of Gene Regulation
9.1 Transcription regulation in Prokaryotes
9.2 Polycistronic genes - lac operon
9.3 Transcriptional termination (attenuation)
9.4 Eukaryotic transcriptional regulation
9.5 Chemical modifications of the DNA
9.6 Regulation via RNA processing and decay
9.7 regulation via translation
9.8 programmed DNA rearrangements in development
© 2006 Jones and Bartlett Publishers
9.4
Eukaryotic gene regulation
transcriptional activator proteins
enhancers
silencers
9.4
Eukaryotic gene regulation
transcriptional activator proteins
binds to upstream DNA
essential for transcription of genes
that are positively regulated
GAL4 fig 9.18
Fig. 9.18. GAL gene
regulation in yeast
© 2006 Jones and Bartlett Publishers
9.4
Eukaryotic gene regulation
enhancers
short sequences
binding sites for transcriptional
activator proteins
most-upstream from start site
9.4
Eukaryotic gene regulation
silencers
short sequences
targets for DNA binding proteins
which block transcription
9.4
Eukaryotic gene regulation
transcriptional complex
aggregate (lots of parts)
binds to promoter ( transcription)
•TFIID
(includes TATA binding protein; TBP)
•TAF’s (TBP associated factors)
•RNA Pol II
fig 9.20
Fig. 9.21. Example of transcriptional activation during Drosophila development
© 2006 Jones and Bartlett Publishers
Fig. 9.20.
Transcriptional
activation by
recruitment
© 2006 Jones and Bartlett Publishers
9.4
Eukaryotic gene regulation
alternative promoters
fig. 9.23
Fig. 9.23. Use of alternative promoters in the gene for alcohol
dehydrogenase in Drosophila
© 2006 Jones and Bartlett Publishers
Chapter 9
Molecular Mechanisms of Gene Regulation
9.1 Transcription regulation in Prokaryotes
9.2 Polycistronic genes - lac operon
9.3 Transcriptional termination (attenuation)
9.4 Eukaryotic transcriptional regulation
9.5 Chemical modifications of the DNA
9.6 Regulation via RNA processing and decay
9.7 regulation via translation
9.8 programmed DNA rearrangements in development
© 2006 Jones and Bartlett Publishers
9.5
Chemically modify the DNA
epigenetic regulation
“in addition to”
changing the DNA,
but not the sequence
methylation
of cytosine
heavy methylation
low transcription
9.5
Chemically modify the DNA
imprinting
maybe later
silencing of specific genes
fig. 9.24
Fig. 9.24. Imprinting genes in chromosomal region 15q11 results in different
neuromuscular syndromes
© 2006 Jones and Bartlett Publishers
Chapter 9
Molecular Mechanisms of Gene Regulation
9.1 Transcription regulation in Prokaryotes
9.2 Polycistronic genes - lac operon
9.3 Transcriptional termination (attenuation)
9.4 Eukaryotic transcriptional regulation
9.5 Chemical modifications of the DNA
9.6 Regulation via RNA processing and decay
9.7 regulation via translation
9.8 programmed DNA rearrangements in development
© 2006 Jones and Bartlett Publishers
9.6
Eukaryotic gene regulation
RNA processing
alternative splicing
fig. 9.25
Fig. 9.25. Alternative
splicing of the primary
transcript of the gene
encoding the alpha chain
of the insulin receptor
© 2006 Jones and Bartlett Publishers
9.6
Eukaryotic gene regulation
RNA persistence
short vs. long-lived mRNA
9.6
Eukaryotic gene regulation
RNAi (interference)
ds RNA is made
cleaved by dicer protein
RISC (RNA-induced silencing complex)
binds to/destroys mRNA
(transferable)
fig. 9.26
Fig. 9.26. Mechanism of RNA interference (RNAi)
© 2006 Jones and Bartlett Publishers
Chapter 9
Molecular Mechanisms of Gene Regulation
9.1 Transcription regulation in Prokaryotes
9.2 Polycistronic genes - lac operon
9.3 Transcriptional termination (attenuation)
9.4 Eukaryotic transcriptional regulation
9.5 Chemical modifications of the DNA
9.6 Regulation via RNA processing and decay
9.7 regulation via translation
9.8 programmed DNA rearrangements in development
© 2006 Jones and Bartlett Publishers
9.7
Eukaryotic gene regulation
translation
small regulatory RNA
base pair with mRNA
antisense RNA
fig. 9.27
on
off
mRNA
Fig. 9.27. Regulation of translation of target mRNAs by the regulatory RNAs.
[After S. Altuvia and E. G.H. Wagner. 2000. Proc. Natl. Acad. Sci. USA 97: 9824. © 2000 National Academy of
Sciences, U.S.A.]
© 2006 Jones and Bartlett Publishers