1. Chapter 16 Lecture Outline
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2. Control of Gene Expression
Chapter 16 2

3. Control of Gene Expression
Controlling gene expression is often accomplished by controlling transcription initiation
Regulatory proteins bind to DNA
May block or stimulate transcription
Prokaryotic organisms regulate gene expression in response to their environment
Eukaryotic cells regulate gene expression to maintain homeostasis in the organism

4. Regulatory Proteins
Gene expression is often controlled by regulatory proteins binding to specific DNA sequences
Regulatory proteins gain access to the bases of DNA at the major groove
Regulatory proteins possess DNA-binding motifs

5. Major groove Minor groove Major groove Minor groove
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Vantage
point
= Hydrogen bond donors
= Hydrogen bond acceptors
= Hydrophobic methyl group
= Hydrogen atoms unable to form hydrogen bonds
DNA molecule 1
Major groove H H N O H N H N G N H N C H
Phosphate N N N H O H
Minor groove
DNA molecule 2
Major groove H H N N H O
CH3
Phosphate N A N H N T H N N
Sugar H O
Minor groove

6. DNA-binding motifs Regions of regulatory proteins which bind to DNA
Helix-turn-helix motif
Homeodomain motif
Zinc finger motif
Leucine zipper motif

7. α Helix (Recognition helix)
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The Helix-Turn-Helix Motif
α Helix
(Recognition helix)
Turn
α Helix
Turn
α Helix
3.4 nm
90° a.

8. 8 a. b. α Helix (Recognition helix) Turn Zipper region α Helix Turn
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The Helix-Turn-Helix Motif
The Leucine Zipper Motif
α Helix
(Recognition helix)
Turn
Zipper region
α Helix
Turn
α Helix
3.4 nm
90° a. b. 8

9. Prokaryotic regulation
Control of transcription initiation
Positive control – increases frequency of initiation of transcription
Activators enhance binding of RNA polymerase to promoter
Effector molecules can enhance or decrease
Negative control – decreases frequency
Repressors bind to operators in DNA
Allosterically regulated
Respond to effector molecules – enhance or abolish binding to DNA

10. Prokaryotic cells often respond to their environment by changes in gene expression
Genes involved in the same metabolic pathway are organized in operons
Induction – enzymes for a certain pathway are produced in response to a substrate
Repression – capable of making an enzyme but does not

11. lac operon Contains genes for the use of lactose as an energy source
-b-galactosidase (lacZ), permease (lacY), and transacetylase (lacA)
Gene for the lac repressor (lacI) is linked to the rest of the lac operon

12. I P CAP-binding site Gene for repressor protein Operator Gene for
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CAP-binding site
Gene for
repressor
protein
Operator
Gene for
permease
Promoter for
lac operon
Promoter
for I gene
Genefor
ß-galactosidase
Gene for
transacetylase P I
CAP
lac O P I Z Y A
Regulatory region
Coding region
lac Control system

13. The lac operon is negatively regulated by a repressor protein
lac repressor binds to the operator to block transcription
In the presence of lactose, an inducer molecule (allolactose) binds to the repressor protein
Repressor can no longer bind to operator
Transcription proceeds
Even in the absence of lactose, the lac operon is expressed at a very low level

14. a. Glucose Low, Inducer Present, Promoter Activated DNA Allolactose
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Glucose Low, Inducer Present, Promoter Activated
DNA
Allolactose
Repressor will
not bind to DNA
CAP-
binding
site
cAMP–CAP binds
to DNA
mRNA
Glucose level
is low cAMP
is high A
CAP
cAMP
cAMP Y Z
cAMP activates
CAP by causing a
conformation change RN A
polymerase is not blocked
and transcription can occur a.

15. blocked by the lac repressor
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Glucose High, Inducer Absent, Promoter Not Activated
Glucose is available
cAMP level is low A
Repressor binds to DNA
CAP does
not bind Y
Effector site is empty,
and there is no
conformation change
RNA polymerase is
blocked by the lac repressor b.

16. Glucose repression
Preferential use of glucose in the presence of other sugars
Mechanism involves activator protein that stimulates transcription
Catabolic activator protein (CAP) is an allosteric protein with cAMP as effector
Level of cAMP in cells is reduced in the presence of glucose so that no stimulation of transcription from CAP-responsive operons takes place
Inducer exclusion – presence of glucose inhibits the transport of lactose into the cell

17. trp operon Genes for the biosynthesis of tryptophan
The operon is not expressed when the cell contains sufficient amounts of tryptophan
The operon is expressed when levels of tryptophan are low
trp repressor is a helix-turn-helix protein that binds to the operator site located adjacent to the trp promoter

18. The trp operon is negatively regulated by the trp repressor protein
trp repressor binds to the operator to block transcription
Binding of repressor to the operator requires a corepressor which is tryptophan
Low levels of tryptophan prevent the repressor from binding to the operator

19. RNA polymerase is not blocked, and transcription can occur
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Tryptophan Absent, Operon Derepressed E
Inactive trp repressor
(No tryptophan present) D
Translation C B A
trp repressor
cannot bind to DNA
mRNA
Enzymes for
tryptophan
synthesis
produced
Operator C B A D E
Gene for
trp repressor
Promoter for
trp operon
RNA polymerase is not blocked,
and transcription can occur a.

20. b. Tryptophan Present, Operon Repressed Tryptophan binds to repressor,
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Tryptophan Present, Operon Repressed
Tryptophan binds to repressor,
causing a conformation change
Tryptophan
Repressor conformation change
allows it to bind to the operator
RNA polymerase is blocked
by the trp repressor, and
transcription cannot occur B A D C
Enzymes for tryptophan
synthesis not produced E
Gene for
trp repressor b.

21. Tryptophan repressor Tryptophan 3.4 nm
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Tryptophan
3.4 nm

22. Eukaryotic Regulation
Control of transcription more complex
Major differences from prokaryotes
Eukaryotes have DNA organized into chromatin
Complicates protein-DNA interaction
Eukaryotic transcription occurs in nucleus
Amount of DNA involved in regulating eukaryotic genes much larger

23. Transcription factors
General transcription factors
Necessary for the assembly of a transcription apparatus and recruitment of RNA polymerase II to a promoter
TFIID recognizes TATA box sequences
Specific transcription factors
Increase the level of transcription in certain cell types or in response to signals

24. Interactions of various factors
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RNA
Polymerase II
Transcribed
region
TAFs B F E
TFIID H
TATA box A
Core promoter

25. Promoters form the binding sites for general transcription factors
Mediate the binding of RNA polymerase II to the promoter
Enhancers are the binding site of the specific transcription factors
DNA bends to form loop to position enhancer closer to promoter

26. Courtesy of Dr.Harrison Echols and Dr. Sydney Kustu
DNA looping
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NtrC (activator)
5 nm
Enhancer
Promoter
RNA polymerase
Bacterial RNA polymerase is loosely bound to the promoter. The activator (NtrC) binds at the enhancer.
ATP
ADP
5 nm
DNA loops around so that the activator comes into contact with the RNA polymerase.
RNA polymerase
Activator
mRNA synthesis
The activator triggers RNA polymerase activation, and transcription begins. DNA unloops.
Courtesy of Dr.Harrison Echols and Dr. Sydney Kustu

27. Enhancers RNA polymerase Transcription factor Transcribed Activator
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Promoter
Enhancer
Activator
Transcription
factor
RNA polymerase
Transcribed
region
TATA box
mRNA synthesis

28. Coactivators and mediators are also required for the function of transcription factors
Bind to transcription factors and bind to other parts of the transcription apparatus
Mediators essential to some but not all transcription factors
Number of coactivators is small because used with multiple transcription factors

29. Transcription complex
Few general principles
Nearly every eukaryotic gene represents a unique case
Great flexibility to respond to many signals
Virtually all genes that are transcribed by RNA polymerase II need the same suite of general factors to assemble an initiation complex

30. Activators General factors B F E RNA polymerase II TFIID H A
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Enhancers
Coding region
Activator
Activators
General factors
These regulatory proteins bind to DNA at distant sites known as enhancers. When DNA folds so that the enhancer is brought into proximity with the initiation complex, the activator proteins interact with the complex to increase the rate of transcription.
Enhancer
Coactivator
TAFs B F
Activator E
RNA polymerase II
TFIID
Coactivators H
These transcription factors stabilize the transcription
complex by bridging activator proteins with the complex. A
General Factors
These transcription factors position RNA polymerase at the start of a protein-coding sequence and then release the polymerase to initiate transcription.

31. Eukaryotic chromatin structure
Structure is directly related to the control of gene expression
DNA wound around histone proteins to form nucleosomes
Nucleosomes may block access to promoter
Histones can be modified to result in greater condensation

32. Methylation once thought to play a major role in gene regulation
Many inactive mammalian genes are methylated
Lesser role in blocking accidental transcription of genes turned off
Histones can be modified
Correlated with active versus inactive regions of chromatin
Can be methylated – found in inactive regions
Can be acetylated – found in active regions

33. Some coactivators have been shown to be histone acetylases
Transcription is increased by removing higher order chromatin structure that would prevent transcription
“Histone code” postulated to underlie the control of chromatin structure

34. Amino acid histone tail Condensed solenoid Acetyl group
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Nucleosomes can block the binding of
RNA polymerase II to the promoter
Amino acid
histone tail
Condensed
solenoid
N-terminus
Addition of acetyl groups to histone
tails remodel the solenoid so that
DNA is accessible for transcription
Acetyl
group
DNA available for transcription

35. Chromatin-remodeling complexes
Large complex of proteins
Modify histones and DNA
Also change chromatin structure
ATP-dependent chromatin remodeling factors
Function as molecular motors
Catalyze 4 different changes in DNA/histone binding
Make DNA more accessible to regulatory proteins

36. Pi ADP + ATP ATP-dependent remodeling factor 1. Nucleosome sliding
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ADP + Pi
ATP
ATP-dependent
remodeling factor
1. Nucleosome sliding
2. Remodeled nucleosome
3. Nucleosome displacement
4. Histone replacement

37. Posttranscriptional Regulation
Control of gene expression usually involves the control of transcription initiation
Gene expression can be controlled after transcription with
Small RNAs
miRNA and siRNA
Alternative splicing
RNA editing
mRNA degradation

38. Micro RNA or miRNA
Production of a functional miRNA begins in the nucleus
Ends in the cytoplasm with a ~22 nt RNA that functions to repress gene expression
miRNA loaded into RNA induced silencing complex (RISC)
RISC is targeted to repress the expression of genes based on sequence complementarity to the miRNA

39. Copyright © The McGraw-Hill Companies, Inc
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RNA Polymerase II
RNA Polymerase II
microRNA gene
microRNA gene
Pri-microRNA
Pri-microRNA
Nucleus
Nucleus
Pre-microRNA
Drosha
Drosha
Pre-microRNA
Exportin 5
Exportin 5
Cytoplasm
Dicer
Mature miRNA
Ago
Ago
RISC
mRNA
Ago
Ago
RISC
mRNA cleavage
Target mRNA
Ago
Ago
Ago
Ago
RISC
RISC 39
Inhibition of translation

40. siRNA RNA interference involves the production of siRNAs
Production similar to miRNAs but siRNAs arise from long double-stranded RNA
Dicer cuts yield multiple siRNAs to load into RISC
Target mRNA is cleaved

41. miRNA or siRNA?
Biogenesis of both miRNA and siRNA involves cleavage by Dicer and incorporation into a RISC complex
Main difference is target
miRNA repress genes different from their origin
Endogenous siRNAs tend to repress genes they were derived from

42. Repeated cutting by dicer
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Exogenous dsRNA, transposon, virus
Repeated cutting
by dicer P P P P P
siRNAs P P P
siRNA
in RISC
Ago +
Ago
RISC
RISC
mRNA
Cleavage of target mRNA

43. Alternative splicing
Introns are spliced out of pre-mRNAs to produce the mature mRNA
Tissue-specific alternative splicing
Same gene makes calcitonin in the thyroid and calcitonin-gene related peptide (CGRP) in the hypothalamus
Determined by tissue-specific factors that regulate the processing of the primary transcript

44. Primary RNA transcript Introns are spliced
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5´cap
3´ Poly- A tail
Primary RNA transcript
Introns are spliced
Exons
Thyroid splicing pattern Hypothalamus splicing pattern
Introns 1 2 3 4 1 2 3 5 6
5´cap
3´ Poly- A tail
5´cap
3´ Poly- A tail
Mature mRNA
Mature mRNA
Calcitonin
CGRP

45. RNA editing
Creates mature mRNA that are not truly encoded by the genome
Involves chemical modification of a base to change its base-pairing properties
Apolipoprotein B exists in 2 isoforms
One isoform is produced by editing the mRNA to create a stop codon
This RNA editing is tissue-specific

46. Initiation of translation can be controlled
Ferritin mRNA only translated if iron present
Mature mRNA molecules have various half-lives depending on the gene and the location (tissue) of expression
Target near poly-A tail can cause loss of the tail and destabilization

47. Copyright © The McGraw-Hill Companies, Inc
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RNA polymerase II
1. Initiation of
transcription .
Transcription is controlled by the frequency of initiation. This involves transcription factors that bind to promoters and enhancers.
2. RNA splicing.
Gene expression
can be controlled
by altering the
rate of splicing in
eukaryotes.
Alternative splicing
can produce
multiple mRNAs
from one gene.
Cut
intron
DNA 3´
3´poly-A tail
5´cap 5´
Primary RNA transcript
Exons
Mature RNA transcipt
Introns

48. Many proteins take part in the translation process, and regulation
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Large
subunit
3. Passage through
the nuclear
membrane.
Gene expression
can be regulated
by controlling
access to or
efficiency of
transport channels.
3´ poly-A tail
Nuclear
pore
mRNA
5´cap
Small
subunit
4. Protein synthesis.
Many proteins take part in the
translation process,
and regulation
of the availability
of any of them alters
the rate of gene
expression by
speeding or slowing
protein synthesis. 3´ 5´

49. Ubiquitin Protein Proteasome
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Ubiquitin
5. RNA interference.
Gene expression
is regulated by
small RNAs. Protein
complexes
containing siRNA
and miRNA target
specific mRNAs for
destruction or inhibit
their translation.
6. Protein degradation. Proteins to be degraded are labeled with ubiquitin, then destroyed by the proteasome.
Protein
RISC
Proteasome

50. Protein Degradation
Proteins are produced and degraded continually in the cell
Lysosomes house proteases for nonspecific protein digestion
Proteins marked specifically for destruction with ubiquitin
Degradation of proteins marked with ubiquitin occurs at the proteasome

51. Pi Ubiquitin Protein to be destroyed ATP ADP +
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Ubiquitin
Protein to be
destroyed
ATP Pi
ADP +
Destroyed by proteolysis

52. Polypeptide fragments
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Degradation
Polypeptide
fragments
Proteasome
ATP
ADP + Pi
Ubiquitination
Ubiquitin
ADP + Pi
ATP
ATP
Targeted
protein
ADP
Ubiquitin ligase