1. Regulation of Gene Expression I
4/15/2017
Scotty Merrell
Department of Microbiology and Immunology
B4140
Regulation of Gene Expression I

2. QUESTIONS 1. Why does the expression of genes need to be regulated?
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QUESTIONS
1. Why does the expression of genes need to
be regulated?
2. Why is it important to study gene regulation?
3. How is the expression of genes regulated?
4. How do we study gene regulation?

3. Bacteria experience different conditions depending on environment
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Pathogenic bacteria:
External reservoir Host
Infection site #1 Infection site #2

4. QUESTIONS 1. Why does the expression of genes need to be regulated?
4/15/2017
QUESTIONS
1. Why does the expression of genes need to
be regulated?
2. Why is it important to study gene regulation?
3. How is the expression of genes regulated?
4. How do we study gene regulation?

5. Pathogenic bacteria produce virulence factors when
they sense they are inside of a host
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ICDDR,B
Vibrio cholerae, the cause of cholera, produces toxin inside
of the host. Understanding regulation of expression of this toxin
is a means of understanding ways to prevent its production.

6. QUESTIONS 1. Why does the expression of genes need to be regulated?
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QUESTIONS
1. Why does the expression of genes need to
be regulated?
2. Why is it important to study gene regulation?
3. How is the expression of genes regulated?
4. How do we study gene regulation?

7. Regulation of Gene Expression
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8. Regulation of Gene Expression
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10. RNA polymerase-promoter interactions
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RNA polymerase-promoter interactions
Some promoters contain UP elements that stimulate transcription
through direct interaction with the C-terminal domains of the
 subunits of the RNA polymerase

11. Arrangement of a subunits on UP elements
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Promoter with a full UP element containing two consensus subsites.
Promoter with an UP element containing only a consensus proximal subsite.
Promoter with an UP element containing only a consensus distal subsite.

12. Genes come in two main flavors:
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Genes come in two main flavors:
Constitutively expressed (transcription initiation
is not regulated by accessory proteins)
Regulated (transcription initiation
is regulated by accessory proteins)
a. Negatively Regulated--Repressor Protein
b. Positively Regulated--Activator Protein

13. Mechanisms of Regulation of Transcription Initiation:
Negative Regulation
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RNA Polymerase

14. Mechanisms of Regulation of Transcription Initiation:
Negative Regulation
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Repressor
Co-repressor
Inactivator
Repressor
Repressor

15. a model for negative regulation
The lac operon
a model for negative regulation
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A bacterium's prime source of food is glucose, since it does
not have to be modified to enter the respiratory pathway. So
if both glucose and lactose are around, the bacterium wants to
turn off lactose metabolism in favor of glucose metabolism.
There are sites upstream of the lac genes that respond to
glucose concentration.
This assortment of genes and their regulatory regions is called
the lac operon.

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19. The lac promoter and operator regions
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20. prevents transcription
The Lac Repressor is constitutively expressed
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Lac Repressor
(monomer)
(tetramer)
Repressor binding
prevents transcription

21. the cell and binds to the Lac repressor, inducing a conformational
When lactose is present, it acts as an inducer of the operon. It enters
the cell and binds to the Lac repressor, inducing a conformational
change that allows the repressor to fall off the DNA. Now the RNA
polymerase is free to move along the DNA and RNA can be made from
the three genes. Lactose can now be metabolized.
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Remember, the
repressor acts
as a tetramer

22. When the inducer (lactose) is removed, the repressor returns to its
original conformation and binds to the DNA, so that RNA polymerase
can no longer get past the promoter to begin transcription. No RNA and
no protein are made.
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Remember, the
repressor acts
as a tetramer

23. How to identify the regulatory elements?
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How to identify the regulatory elements?
1. Mutation in the regulatory circuit may either abolish
expression of the operon or cause it to occur without
responding to regulation.
2. Two classes of mutants:
A. Uninducible mutants: mutants cannot be expressed at all.
B. Constitutive mutants: mutants continuously express
genes that do not respond to regulation.
3. Operator (lacO): cis-acting element
Repressor (lacI): trans-acting element

24. Definitions: cis-configuration: description of two sites on the same
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cis-configuration: description of two sites on the same
DNA molecule (chromosome)
or adjacent sites.
cis dominance: the ability of a gene to affect genes
next to it on the same DNA molecule
(chromosome), regardless of the nature
of the trans copy. Such mutations exert
their effect, not because of altered
products they encode, but because of a physical
blockage or inhibition of RNA transcription.
trans-configuration:description of two sites on different
DNA molecules (chromosomes)
or non-contiguous sites.

25. Constitutive mutants: do not respond to regulation.
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Would this be a cis-dominant or recessive mutation?

26. Constitutive mutants can be recessive
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Constitutive mutants can be recessive

27. Constitutive mutants can also be dominant if the mutant allele produces a “bad” subunit, which is not only itself unable to bind to operator DNA, but is also able to act as part of a tetramer to prevent any “good” (wild type LacI) subunits from binding.
4/15/2017 Pi
lacI- P O
lacZ
lacY
lacA X
mRNA
mRNA
et al.
mRNA
lacI +

28. Think about how you could determine whether a mutation was dominant or
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Think about how you could determine
whether a mutation was dominant or
recessive.

29. Questions about negative
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Questions about negative
Regulation of lac ?

30. Mechanisms of Regulation of Transcription Initiation:
Positive Regulation
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RNA Polymerase

31. Mechanisms of Regulation of Transcription Initiation:
Positive Regulation
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RNA Polymerase
Activator

32. a model for positive regulation
The lac operon
a model for positive regulation
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When levels of glucose (a catabolite) in the cell are high, a molecule called cyclic AMP is inhibited from forming. So when glucose levels drop, more cAMP forms. cAMP binds to a protein called CAP (catabolite activator protein), which is then activated to bind to the CAP binding site. This activates transcription, perhaps by increasing the affinity of the site for RNA polymerase. This phenomenon is called catabolite repression, a misnomer since it involves activation, but understandable since when it was named, it seemed that the presence of glucose repressed all the other sugar metabolism operons.

33. CAP --- a positive regulator
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CAP --- a positive regulator
1. Catabolite repression: the decreased expression of many bacterial operons that results from addition of glucose. Also known as “glucose effect” or “glucose repression”.
2. E. coli catabolite gene activator protein (CAP; also known as CRP, the cAMP receptor protein).
3. CAP-cAMP activates more than 100 different promoters, including promoters required for utilization of alternative carbohydrate carbon sources such as lactose, galactose, arabinose, and maltose.

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35. How does glucose reduce cAMP level?
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How does glucose reduce cAMP level?
PTS
Glucose
Glucose-6-P
IIAGlc-P
IIAGlc
OUT IN
PTS - phosphoenolpyruvate-dependent carbohydrate
phosphotransferase system
IIAGlc - glucose-specific IIA protein, one of the
enzymes involved in glucose transport.
1. IIAGlc-P activates adenylate cyclase.
2. Glucose decreases IIAGlc-P level,
thus reducing cAMP production.
3. Glucose also reduces CAP level:
crp gene is auto-regulated by
CAP-cAMP.

36. Activation of expression of the lac operon
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37. E. coli CAP (CRP) --- 209 amino acids
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E. coli CAP (CRP) amino acids
DNA-binding
Helix-turn-helix
Dimerization and cAMP-binding
1-139
NH2-
AR1
-COOH
His19
His21
Glu96
Lys101
AR2

38. Transcription activation by CAP at class I CAP-dependent promoters
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(-62)
Transcription activation:
Interaction between the AR1 of the downstream CAP subunit and one copy of aCTD.
The AR1-aCTD interaction facilitates the binding of aCTD to the DNA downstream of CAP.
Possibly, interaction between same copy of aCTD and the s70 bound at the –35 element.
4. The interaction between the second aCTD and CAP is unclear.
The result: increasing the affinity of RNAP for promoter DNA, resulting in an
increase in the binding constant KB, for the formation of the RNAP-promoter closed complex

39. Transcription activation by CAP at class I
CAP-dependent promoters (cont.)
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(-103, -93, -83, or –72)
Transcription activation:
Possibly, the second copy of aCTD may interact with the DNA downstream of CAP, and
may interact with the s70 bound at the –35 element.
Results: increasing the affinity of RNAP for promoter DNA, resulting in an
increase in the binding constant KB, for the formation of the RNAP-promoter closed complex

40. Transcription activation by CAP at class II
CAP-dependent promoters (cont.)
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(-42)
Transcription activation:
Interaction between the AR1 of the upstream CAP subunit and one copy of aCTD
(either aCTDI or aCTDII, but preferentially aCTDI). The AR1-aCTD
interaction facilitates the binding of aCTD to the DNA upstream of CAP.
Results: increase in the binding constant KB, for the formation of the RNAP-promoter
closed complex
Interaction between the AR2 of the downstream CAP subunit and aNTDI.
Result: increase the rate constant, kf, for isomerization of closed complex to open complex.

41. Transcription activation by CAP at class III CAP-dependent promoters
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(-103 or –93)
(-62)
Transcription activation:
Each CAP dimer functions through a class I mechanism with AR1 of the downstream subunit of each CAP dimer interacting with one copy of aCTD
Results: synergistic transcription activation

42. Transcription activation by CAP at class III
CAP-dependent promoters (cont.)
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(-103, -93, or -83)
(-42)
Transcription activation:
The upstream CAP dimer functions by a class I mechanism, with AR1 of the downstream subunit interacting with one copy of aCTD; the downstream CAP dimer functions by a class II mechanism, with AR1 and AR2 interacting with the other copy of aCTD and aNTD, respectively.
Results: synergistic transcription activation

43. No lactose inside the cells!
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(a) Glucose present (cAMP low); no lactose;
(b) Glucose present (cAMP low); lactose present
lacI Pi P O
lacY
lacZ
lacA
Repressor
monomer
tetramer
mRNA X
No lactose inside the cells!
(inducer exclusion)!
Repressor
monomer
tetramer
mRNA
Inducer
High level
of mRNA X
Inactive
repressor
High
(c) No glucose (cAMP high); lactose present
cAMP
CAP
Glucose effect on
the E. coli lac operon

44. No lactose inside the cells!
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(a) Glucose present (cAMP low); no lactose;
(b) Glucose present (cAMP low); lactose present
lacI Pi P O
lacY
lacZ
lacA
Repressor
monomer
tetramer
mRNA X
No lactose inside the cells!
(inducer exclusion)!
Repressor
monomer
tetramer
mRNA
Inducer
High level
of mRNA X
Inactive
repressor
High
(c) No glucose (cAMP high); lactose present
cAMP
CAP
Glucose effect on
the E. coli lac operon

45. Inducer exclusion: How does it work?
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Uptake of glucose dephosphorylates enzyme IIglc.
Dephosphorylated enzyme IIglc binds to and inhibits lactose permease.
Inhibition of lactose permease prevents lactose from entering the cell.
Hence, the term inducer exclusion.

46. Questions about positive regulation
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Questions about positive regulation
of the lac operon?

47. Dual positive and negative control of transcription initiation:
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Dual positive and negative control
of transcription initiation:
the E. coli ara operon

48. The E. coli L-arabinose operon
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The E. coli L-arabinose operon + +

49. AraC exists in two states
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Arabinose P1 P2
Antiactivator
Activator
Arabinose

50. AraC acts as a positive or negative regulator based on its conformational state and binding affinity for various sites in the two promoter regions.
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AraC encodes the regulator
AraO1 and AraO2 encode operators
CAP is a CAP binding site
AraI is an additional regulatory region
AraBAD are the structural genes

51. In the absence of arabinose, the P1 form of AraC binds AraO2 and
AraI to prevent any P2 form from binding and activating expression
--this is anti-activation, not repression!
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No arabinose
+ arabinose
In the presence of arabinsose, AraC shifts to the P2 form and binds
AraI and acts to activate transcription.
If AraC concentration becomes too high, AraC will also bind to AraO1
and repress its own expression.
Therefore AraC is an Activator, Repressor and Anti-activator!!

52. The domain structure of one subunit of the dimeric AraC protein
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The regulatory regions of the PC and PBAD promoters
The domain structure
of one subunit of the
dimeric AraC protein

53. The PC and PBAD Regions in the presence or absence of arabinose
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The PC and PBAD Regions in the presence
or absence of arabinose
+ L-arabinose

54. Hypothetical model of the activation of the PBAD promoter
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Hypothetical model of the activation of the PBAD promoter
PBAD – class II promoter
Possible interactions: between the aCTD of RNAP
and the CAP protein and AraC protein and DNA

55. Strategies for Understanding Regulation
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Strategies for Understanding Regulation
1. Find mutations that render the regulation uninducible or constitutive.
2. Decide by performing a complementation test if the mutants are dominant or
recessive.
3. If they are recessive, decide if the system is regulated by repression or by
activation. A recessive mutated activator has most likely lost function: the
system will become uninducible. A recessive mutated repressor has also lost
function, but now the system will show constitutive expression.
4. Decide if the elements of the system act in cis or in trans to each other: are
they proteins or DNA binding sites?
5. Construct a model.

56. Questions about ara regulation?
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Questions about ara regulation?

57. A. Transcriptional control 1. Transcription initiation a) Positive
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Regulatory mechanisms used to control gene expression
A. Transcriptional control
1. Transcription initiation
a) Positive
b) Negative
2. Transcription termination
Attenuation
B. Translational control
1. Positive
2. Negative
C. Post-translational control--Proteolysis

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59. Transcription termination players: and sometimes the Rho (r) factor
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Transcription termination players:
Termination sequence
RNA polymerase
and sometimes the Rho (r) factor
RNAP A B C D X
Promoter
Operon of 4 genes
Terminator

60. Two major types of Terminator Sequences
1. Rho-independent
2. Rho-dependent
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Rho-independent
terminator
Rho-independent
terminator
Rho-dependent
terminator

61. Premature termination of transcription
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Attenuation:
Premature termination of transcription
of operons for amino acid biosynthesis
(trp, his, leu, etc.)
Bacterial version of
Supply and Demand
Relies on coupled transcription and translation and
RNA secondary structure

62. Organization of Tryptophane Biosynthesis Genes
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Organization of Tryptophane Biosynthesis Genes
End product of the pathway
The trp leader mRNA encodes the LEADER PEPTIDE
MetLysAlaIlePheValLeuLysGlyTrpTrpArgThrSer
5’-AUGAAAGCAAUUUUCGUACUGAAAGGUUGGUGGCGCACUUCC U
CCCAUAGACUAACGAAAUGCGUACCACUUAUGUGACGGGCAAAG A
GCCCGCCUAAUGAGCGGGCUUUUUUUUGAACAAAAUUAGAGA-3’ 1 3 2 4

63. mRNA forms secondary structures
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Pre-emptor
2 and 3 form the
Pre-emptor, which prevents
Terminator formation
3 and 4 form a
Rho-independent
terminator
Two possible alternative structures can form
2 is complementary to 1 and 3
3 is complementary to 2 and 4
Adapted from

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Tryptophan absent
Tryptophan present

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67. Attenuation can also occur at the level of Protein-RNA interaction:
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Attenuation can also occur at the level of
Protein-RNA interaction:
Regulation of the trp operon in Bacillus

68. Model of trp transcriptional control Binding of activated TRAP
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Binding of
activated TRAP
in the leader
peptide
results in the
formation of a
terminator
structure

69. Transcription of genes to produce mRNA
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Take home message:
Transcription of genes to produce mRNA
can be controlled at the level of
initiation and/or termination

70. 4/15/2017
STOP