1. Regulation of Gene Expression in Bacteria
MB 206 : Module 1 - B
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2. This diagram is for eukaryote
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3. Regulation of Gene Expression
A cell contains the entire genome of an organism– ALL the DNA.
Gene expression = transcribing and translating the gene
Regulation allows an organism to selectively transcribe (and then translate) only the genes it needs to.
Genes expressed depend on
the type of cell
the particular needs of the cell at that time.

4. Principles of Gene Regulation
In genetics, constitutive refers to a gene product made all the time.
In the absence of the activator or the repressor, RNA polymerase transcribes the gene constitutively
Constitutive
Inducible
A gene is expressed in higher level under influence of some signal
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5. How Are Genes Regulated?
Genes located in coherent packages called operons
operons has 4 parts
regulatory gene - controls timing or rate of transcription
promoter - starting point
operator - controls access to the promoter by RNA polymerase
structural genes
NOTE = operons regulated as units
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7. Gene Regulation in Prokaryotes
Prokaryotes organize their genome into operons
Operon = a group of related genes
One promoter sequence at the very beginning
All of the genes will be transcribed together (in one long strand of RNA.

8. Principle of Gene Regulation
RNA polymerase binds to DNA at promoters.
Transcription initiation is regulated by proteins that bind to or near promoters.
Repression of a repressible gene: (i.e., negative regulation) repressors (vs activitors) bind to operators of DNA.
Repressor is regulated by an effector, usually a small molecules or a protein, that binds and causes a conformational change.
Activitor binds to DNA sites called
enhancer to enhance the RNA
polymerase activity.
(i.e., [positive regulation)
Induction of an inducible gene, e.g., heat-shock genes.
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9. General organization of an inducible gene

10. Regulation of Genes Transcription Factor (Protein) RNA polymerase DNA
Regulatory Element
Gene

11. Regulation of Genes
New protein
RNA polymerase
Transcription Factor
DNA
Regulatory Element
Gene

12. Gene Expression How much protein is in a cell (and active)??
Some gene products are necessary only some of the time
Response to life events (eg. Reproduction)
Response to environmental changes (eg. Temperature)
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13. Most genes are not expressed at a particular time
Not all of the genes in a bacteria will be expressed at the same time.
Even in some of the smallest bacteria, about 500 different genes exists
Of the 4279 genes in E. coli , only about 2600 (~60%) are expressed in standard laboratory conditions.
Only about 350 genes are expressed at more than 100 copies (i.e. molecules!) per cell, making up 90% of the total protein.
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16. Possible target in control of gene expression

17. Topics: Lac Operon (Negative control & Catabolic repression)
Tryptophan Operon (Positive control)
Histidine Operon (Attenuator)
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18. ORGANISM Size of genome (bp) number of genes
Comparison of genomes of various organism
ORGANISM
%coding
Size of genome (bp)
number of genes
Escherichia coli
90%
4,639,221 bp
4288
Mycoplasma genitalium
88%
580,073 bp
468
Haemophilus influenzae
86%
2,087,778 bp
1,662
Methanococcus jannaschii
85%
1,660,000 bp
1,997
Synechocystis sp.
(PCC 6803)
80%
3,570,000 bp
3,168
Saccharomyces cerevisiae
~75%
13,000,000 bp
6,275
Humans
~2%
3,000,000,000 bp
70,000 (?)
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19. Mus musculus (house mouse) 40 Drosophila melanogaster (fruit fly) 8
Diploid numbers of some commonly studied organisms (as well as a few extreme examples)
Homo sapiens (human) 46
Mus musculus (house mouse) 40
Drosophila melanogaster (fruit fly) 8
Caenorhabditis elegans (microscopic roundworm) 12
Saccharomyces cerevisiae (budding yeast) 32
Arabidopsis thaliana (plant in the mustard family) 10
Xenopus laevis (South African clawed frog) 36
Canis familiaris (domestic dog) 78
Gallus gallus (chicken)
Zea mays (corn or maize) 20
Muntiacus reevesi (the Chinese muntjac, a deer) 23
Muntiacus muntjac (its Indian cousin) 6
Myrmecia pilosula (an ant) 2
Parascaris equorum var. univalens (parasitic roundworm)
Cambarus clarkii (a crayfish)
200
Equisetum arvense (field horsetail, a plant)
216
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20. Genes in E.coli
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21. E.coli genes expressed A total of 4288 genes in Escherichia coli
genes found under standard laboratory growth conditions
protein spots detected under 2-D protein gels
- 350 proteins in high amount, the rest are very low amounts
Majority of the genes are likely to be expressed transiently, in small amounts during DNA replication, and then remain silent (unexpressed) until the next round of DNA synthesis

22. Why is there a need to control gene expression?
1) Prevent energy wastage
2) Ensure only necessary proteins are made according to the requirement for cells growth.
Small portion of DNA in cell used for genetic message (mRNA), the rest for regulatory purposes.
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23. Gene Regulation in bacteria
How do single-celled prokaryotes like E. coli know how to respond to their environments?
Each environmental cue generates a specific response, with specific proteins and reactions.
eg.
A bacterium can use different sources of Nitrogen
- incorporate N2 gas from the air
- incorporate ammonia from their surroundings or
- from amine group of an amino acid like glutamine (easier and less energy)
These processes involve very different enzymes. Presence of glutamine, the cell will turn off synthesis of enzymes for fixing N2
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24. How can the cell "turn off" the synthesis of proteins from its DNA?
Expression of genes in microbes is often regulated by intracellular or environmental conditions.
Many different types of regulation
Most genes are regulated at the point of transcription initiation – early intervention control
Regulation at different stage of gene expression:
- transcription initiation or termination – no production of mRNA
- mRNA stability or translation into protein.
- protein modification and stability - prevent wasteful reactions.
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25. DNA
Transcription
mRNA
Translation
Protein
Gene regulation can occur at any place along the flow of information from DNA to RNA to protein:
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26. Different forms of gene regulation
a. Regulation by DNA Replication (default)
b. Transcriptional Regulation by different s-factors.
c. Negative Regulation of Gene Expression
d. Positive Control of Gene Regulation
e. Alternative splicing of RNA (almost exclusively for eukaryotes)
f. Post-transcriptional regulation
- termination of transcription
- translation control message stability protein stability
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27. E.coli RNA Polymerase subunits
Gene
Mass
KDa
Use
-35 Sequence
separation
-10 Sequence
rpoA 40
a subunit -
rpoB
155
b subnit
rpoC
160
b' subunit
rpoD 70
s70
General
TTGACA
16-18 bp
TATAAT
rpoN 54
s54
Nitrogen
CTGGNA
6 bp
TTGCA
rpoS 38
s38
Stationary
not known
rpoH 32
s32
Heat shock
CCCTTGAA
13-15 bp
CCCGATNT
fliA 28
s28
Flagellar
CTAAA
15 bp
GCCGATAA
rpoE 24
s24
High temp.
heat shock
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28. Transcription regulation by s-factors
s70 - RpoD “normal” s-factor s54 - RpoN Nitrogen response s38 - RpoS Stationary phase s32 - RpoH Heat shock response s28 - FliA Flagellar genes regulation s24 - RpoE Heat shock high temp.
Approx: copies of RNAP holoenzyme/ cell
For bacteria growing in "log phase":
~600 copies of RpoD (s70)
~200 copies of RpoS (s38)
[RpoS] increases to ~600 copies per cell in stationary phase or osmotic shock.
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29. Operon and Regulon An operon
- consists of a set of genes expressed coordinately & transcribed
as a single unit
- Specific regulation (positive / negative) can induce or repress a
particular gene or operon
- contains both a regulatory & a message region.
- Regulatory / control region at the 5’ side of the gene & codes for a
protein (message region).
Regulon
- comprise of global regulation affecting a set of operons.
- All operons in the regulon are coordinately controlled by the
same regulatory mechanism.
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30. Operons-the basic concept of Prokaryotic Gene Regulation
Regulated genes can be switched on and off depending on the cell’s metabolic needs
Operon : a regulated cluster of adjacent structural genes, operator site, promotor site, and regulatory gene(s)

31. Repressible vs. Inducible Operons two types of negative gene regulation
Repressible Operons
Genes are initially ON
Anabolic pathways
End product switches off its own production
Inducible Operons
Genes are initially OFF
Catabolic pathways
Switched on by nutrient that the pathway uses

32. lac: an inducible operon

33. The lac Operon of E. coli
1. Growth and division genes of bacteria are regulated genes. Their expression is controlled by the needs of the cell as it responds to its environment with the goal of increasing in mass and dividing. 2. Genes that generally are continuously expressed are constitutive genes (housekeeping genes). Examples include protein synthesis and glucose metabolism. 3. All genes are regulated at some level, so that as resources dwindle the cell can respond with a different molecular strategy. 4. Prokaryotic genes are often organized into operons that are cotranscribed. A regulatory protein binds an operator sequence in the DNA adjacent to the gene array, and controls production of the polycis-tronic (polygenic) mRNA. 5. Gene regulation in bacteria and phage is similar in many ways to the emerging information about gene regulation in eukaryotes, including humans. Much remains to be discovered; even in E. coli, one of the most closely studied organisms on earth, 35% of the genomic ORFs have no attributed function.
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34. The lac Operon of E. coli
Animation: Regulation of Expression of the lac Operon Genes
1. An inducible operon responds to an inducer substance (e.g., lactose). An inducer is a small molecule that joins with a regulatory protein to control transcription of the operon.
2. The regulatory event typically occurs at a specific DNA sequence (controlling site) near the protein-coding sequence (Figure 16.1).
3. Control of lactose metabolism in E. coli is an example of an inducible operon.
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35. Lac Operon Transcription is “OFF”
When there is no lactose that needs to be digested
lacI repressor is in active form  binds to operator  blocks RNA Polymerase  no transcription

36. Lac Operon Transcription is “ON”
When there is lactose that needs to be digested
Lactose binds to lacI repressor  inactivates it
RNA Polymerase is able to bind to promoter  transcribe genes

37. Negative Regulation of Gene Expression
By default, the gene is usually
switched ON.
Binding of a REPRESSOR will switch
the gene OFF.
Most common regulation in BACTERIA
Often this is found as AUTOREGULATION - where too much of the gene product inhibits further transcription - usually this is through binding to the upstream promoter control region.
A good "classic" example is the E.coli lac operon.
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38. Positive control of Regulation
By default, the gene is usually
switched OFF.
Binding of a ACTIVATOR will switch
the gene ON. (often transcriptional activators / factors
bind and bend DNA upstream of the promoter.)
Most common in EUKARYOTES
Some promoters are not very functional in the absence of a transcriptional activator protein(s).
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39. Lac Operon
Lactose metabolism occurs when the environment contains lactose.
Enzymes required for lactose degradation are TURNED ON.
beta-galactosidase (lac Z)
- enzyme hydrolyzes the bond between glucose & galactose.
Lactose Permease (Lac Y)
- enzyme spans the cell membrane
- transports lactose into the cell from the outside environment.
- Membrane is otherwise essentially impermeable to lactose.
Thiogalactoside transacetylase (LacA)
- The function of this enzyme is not known.
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40. Lactose metabolism
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42. Regulatory elements in the Lac Operon
Element Function
Operator (LacO) binding site for repressor
Promoter (LacP) binding site for RNA polymerase
Repressor (LacI) codes for lac repressor protein
Binds to DNA at operator and blocks binding of RNA polymerase at promoter
Pi promoter for Lac I
CAP binding site for cAMP/CAP complex
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43. Glucose or Lactose?
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 & lactose are around, the bacterium will
to turn off lactose metabolism in favor of glucose metabolism.
There are sites upstream of the Lac genes that respond
to different glucose concentrations.
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44. Presence of inducer - lactose
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45. Regulation of Lac operon - depending on availability of lactose or glucose
Low levels of Glucose /
Catabolite repression
Absence of lactose
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46. CAP will bend the DNA around it and facilitate transcription initiation by RNA polymerase.
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47. Regulation of Lac Operon
When lactose is present, it acts as an inducer of the operon. Lactose enters the cell and binds to the Lac repressor, inducing a conformational change preventing the repressor from binding to the operator. This allows the RNA polymerase binding at the promoter to proceed with transcription of mRNA (LacZ, LacY & LacA) and production of enzymes for the metabolism of lactose.
When the inducer (lactose) is removed, the repressor returns to its original conformation and binds to operator, blocking the RNA polymerase from proceeding with transcription of mRNA, thus no protein is made.
The lac operon is always primed for transcription upon the addition of lactose.
When levels of glucose (a catabolite) in the cell are high, formation of cyclic AMP is inhibited. But 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, by increasing the binding affinity of RNA polymerase to promoter. This is called catabolite repression, a misnomer since it involves activation, but understandable since it seemed that the presence of glucose repressed all the other sugar metabolism operons.
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48. The Tryptophan Operon (Positive regulation)
Trp operon contains the Tryptophan biosynthetic genes.
Trp repressor protein can bind to the operator of Trp operon
When tryptophan is high, it binds to the repressor and induces a change so that the repressor can now bind to DNA.
When tryptophan are low in the cell, tryptophan falls off the repressor, and the repressor goes back to its original conformation, losing its ability to bind to the DNA. RNA polymerase binds to the promoter and transcription proceeds, making tryptophan biosynthetic genes and replenishing the cell's supply of tryptophan.
This type of feedback inhibition of transcription is very common. ribosomal RNA can also act to repress their own synthesis.
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50. Repressible Operon: Trp Operon
Repressible Operon = Operon that is usually “ON” but can be inhibited
The Trp Operon
example of a repressible operon
Genes that code for enzymes needed to make the amino acid tryptophan

51. TrpR Gene TrpR gene is the regulatory gene for the Trp operon
Found somewhere else on the genome
NOT part of the Trp operon
TrpR gene codes for a protein = TrpR repressor
TrpR gene is transcribed and translated separately from the Trp operon genes.

52. TrpR Repressor Repressor protein is translated in an inactive form
Tryptophan is called a corepressor
When tryptophan binds to the TrpR repressor, it changes it into the active form

53. Operator Region
There is also an operator region of DNA in the Trp Operon
Just after the promoter region
The TrpR Repressor can bind to the operator if it’s in the active form

54. Trp Operon Transcription is “ON”
Occurs when there is no tryptophan available to the cell.
Repressor is in inactive form (due to the absence of tryptophan)
RNA Polymerase is able to bind to promoter and transcribe the genes.

55. Trp Operon Transcription is “OFF” Occurs when tryptophan is available
Tryptophan binds to the TrpR repressor  converts it to active form
TrpR protein binds to operator  blocks RNA Polymerase  no transcription

56. trp: a repressible operon

57. Question…
Under what conditions would you expect the trp operon to go from “OFF” to “ON” again?
When there is no longer tryptophan available– all of it has been used up

58. The Histidine Operon (An Attenuator)
The histidine operon functions in a slightly different way.
At the beginning of the operon there is a leader coding region
AUG-AAA-CGC-GUU-CAA-UUU-AAA-CAC-CAC-CAU-CAU-CAC-CAU-CAU-CCU- GAC
Met-Thr-Arg-Val-Gln-Phe-Lys-His-His-His-His-His-His-His-Pro- Asp
When transcription begins, the RNA comes of the DNA and ribosomes hop onto it to start translation.
Low amount of histidine in the cell:
- the ribosome stalls because no aminoacyl tRNA's charged with histidine.
- this leaves a long stretch of RNA (for RNA ploymerase is still transcribing
it) with no ribosomes bound to it.
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59. The Histidine Operon (An Attenuator)
High amount of histidine in the cell:
- the ribosome is not stalled
- the leader sequence in RNA allows it to form a terminator loop
(attenuation site), at which point the RNA is cleaved
- RNA polymerase stops transcribing the genes
- the terminator only functions when the ribosome is not stalled.
Many amino acid synthetic operons are also controlled by some form of attenuation. The tryptophan operon has both attenuation control and repressor control.
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61. Methods for Studying Regulation
Possible mutations in various elements of the Lac operon, give rise to mutants
How to study the lac operon? Tools used:
IPTG (isopropyl-beta-D-thiogalactoside)
- a molecule analogue to lactose, binds to the Lac repressor (Lac I).
- used as a gratuitous inducer to induce Lac operon
- but not a substrate for the lactose metabolism genes.
Spectrophotometer quantification of B-galactosidase activity.
- Quantify amount of mRNA made (coding lacZ, lacY, and lacA)
- B-galactosidase can cleave a colourless substrate called ONPG into a
yellow product , ONP - quantitated by spectrophotometer.
- The degree of yellowness - indicates enzyme activity or amount of
transcription of mRNA or the activity of lac operon.
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62. Methods for Studying Regulation
Different types of gene expression:
- Constitutively ( c ) expressed gene is never turned off, it is
making mRNA and protein all the time.
- Inducible gene can be turned on by an inducer.
- Uninducible gene is never turned on. DNA binding site is
mutated preventing binding by an inducer.
- Super-repressor ( s ) always represses, regardless
of its regulation. eg. a Lac I (s) mutant always represses the
promoter whether or not lactose is present.
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63. Cis or Trans-regulation
DNA element 1 and DNA element 2
How to determine whether DNA element 1 is acting in cis or in trans to one another ?
Test: insert a piece of DNA carrying DNA element 1 into a cell that already has a copy of mutated DNA element 1 adjacent to DNA element 2.
A) Observation: the cell recovers it’s function.
Conclusion: The inserted element can complement or replace the function of the mutated element 1, it can be said to be trans acting, since it must diffuse off a plasmid or from another site in the DNA in order to be functional. This, therefore involves a diffusible protein.
B) Observation: the cell does not regains it’s function
Conclusion: the two functional pieces of DNA must be adjacent to each other to be functional (cis acting),
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65. BLA OPERON - Control of gene expressing resistance to Penicillin
Usually present on a plasmid in S aureus;
its function is B-lactam-induced production of penicillinase.
Bla operon composed of 3 genes:
blaZ = codes for a penicillin-hydrolysing enzyme (penicillinase)
blaR1 & bla l = transcription regulator genes
When penicillin is in the environment, membrane-bound signal transducer protein BlaR1 recognises it and transmits the signal to the cytoplasm.
Repressor protein Bla I, which binds near to the promoter of blaZ preventing its transcription, is cleaved off.
blaZ is transcribed efficiently to produce penicillinase.
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66. Negative regulation:
Substrate induction

67. Positive
regulation of
the lac
operon

68. Positive Gene Regulation
In the lac operon there are other molecules to further stimulate transcription.
Lactose will only be digested for energy when there isn’t much glucose around
When glucose levels are low, level of cAMP molecule builds up

69. cAMP and CAP CAP = regulatory protein that binds to cAMP
CAP is inactive unless cAMP binds to it

70. Positive gene regulation
If there isn’t much glucose high levels of cAMP
CAP and cAMP bind  CAP can bind to the promoter  stimulates RNA Polymerase to bind

71. Positive gene regulation
When glucose levels rise again, cAMP levels will drop  no longer bound to CAP
CAP can’t bind to promoter  transcription slows down

72. Positive gene regulation
The lac operon is controlled on 2 levels:
Presence of lactose determines if transcription can occur
CAP in the active form determines how fast transcription occurs

73. Lac Operon Operon is on and enhanced Operon is ready to be enhanced
Glucose?
Low: cAMP high, CRP attaches
Lactose?
Low: Repressor attached
No Food
Glucose?
Low: cAMP high, CRP attaches
Lactose?
Low: Repressor removed
Milk
Operon is on and enhanced
Operon is ready to be enhanced
but is shut off

74. Lac Operon Operon is off and un-enhanced Operon is on but un-enhanced
Glucose?
High: cAMP low, CRP off
Lactose?
Low: Repressor removed
Milkshake
Glucose?
High: cAMP low, CRP off
Lactose?
Low: Repressor attached
Powerade
Operon is off and un-enhanced
Operon is on but un-enhanced
POWERade is a drink manufactured by The Coca-Cola Company.

75. Lac Operon No food ready to be enhanced but off Milk
enhanced transcription
Milkshake
un-enhanced transcription
Powerade
off and un-enhanced

76. Induction by negative or positive control
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77. Negative regulation: end-product repression

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80. Question… What is the benefit of organizing the genome into operons?
It’s more efficient – transcribe everything you need for a process at once.

81. Do all operons have operator regions?NO
There are some genes that always need to be transcribed  they do not need to have operators to regulate them in this manner.
Ex. genes that participate in cellular respiration

82. Thank U