Download - MB 206 : Module 1 - B
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Regulation of Gene Expression in Bacteria
This diagram is for eukaryote
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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.
Principles of Gene Regulation
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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 unitsAngelia Teo Jan 09
Angelia Teo Jan 09
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.
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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General organization of an inducible gene
Regulation of Genes
Gene
RNA polymerase
Transcription Factor(Protein)
Regulatory ElementDNA
Regulation of Genes
Gene
RNA polymeraseTranscription Factor
Regulatory Element
DNA
New protein
Gene Expression
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How much protein is in a cell (and active)??
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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Possible target in control of gene expression
Topics:
Lac Operon (Negative control & Catabolic repression)
Tryptophan Operon (Positive control)
Histidine Operon (Attenuator)
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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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Comparison of genomes of various organism
Diploid numbers of some commonly studied organisms
(as well as a few extreme examples)Homo sapiens (human) 46 Mus musculus (house mouse) 40Drosophila melanogaster (fruit fly) 8Caenorhabditis elegans (microscopic roundworm) 12
Saccharomyces cerevisiae (budding yeast) 32Arabidopsis thaliana (plant in the mustard family) 10
Xenopus laevis (South African clawed frog) 36Canis familiaris (domestic dog) 78Gallus gallus (chicken) 78Zea mays (corn or maize) 20Muntiacus reevesi (the Chinese muntjac, a deer) 23
Muntiacus muntjac (its Indian cousin) 6Myrmecia pilosula (an ant) 2Parascaris equorum var. univalens (parasitic roundworm) 2
Cambarus clarkii (a crayfish) 200
Equisetum arvense (field horsetail, a plant) 216Angelia Teo Jan 09
Genes in E.coli
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E.coli genes expressed
A total of 4288 genes in Escherichia coli - 2600 genes found under standard laboratory
growth conditions - 2100 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
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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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 Angelia Teo Jan 09
How can the cell "turn off" the synthesis of proteins from its DNA?
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Gene regulation can occur at any place along the flow of information from DNA to RNA to protein:
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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 Regulatione. 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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E.coli RNA Polymerase subunits
Gene MassKDa Use -35 Sequence separation -10 Sequence
rpoA 40 subunit - - -rpoB 155 subnit - - -rpoC 160 ' subunit - - -
rpoD 70 70
General TTGACA 16-18 bp TATAAT
rpoN 54 54
Nitrogen CTGGNA 6 bp TTGCA
rpoS 38 38
Stationary not known not known not known
rpoH 32 32
Heat shock CCCTTGAA 13-15 bp CCCGATNT
fliA 28 28
Flagellar CTAAA 15 bp GCCGATAA
rpoE 2424
High temp.heat shock
not known not known not known
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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: 1500 - 2000 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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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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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)
Repressible vs. Inducible Operonstwo 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
The lac Operon of E. coli1. 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.
台大農藝系 遺傳學 601 20000Chapter 16 slide 33
The lac Operon of E. coliAnimation: Regulation of Expression of the lac Operon
Genes1. 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.
台大農藝系 遺傳學 601 20000Chapter 16 slide 34
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
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
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. Angelia Teo Jan 09
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). Angelia Teo Jan 09
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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Lactose metabolism
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Regulatory elements in the Lac Operon
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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 ICAP binding site for cAMP/CAP complex
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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Presence of inducer - lactose
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Low levels of Glucose / Catabolite repression Absence of lactose
Regulation of Lac operon - depending on availability of lactose or glucose
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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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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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Angelia Teo Jan 09
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
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.
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
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
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.
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
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
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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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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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) - -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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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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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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BLA OPERON- Control of gene expressing resistance to Penicillin
Usually present on a plasmid in S aureus; its function is -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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Negative regulation:Substrate induction
Positive regulation of the lac operon
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
cAMP and CAP
CAP = regulatory protein that binds to cAMP
CAP is inactive unless cAMP binds to it
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
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
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
Lac Operon
No Food Milk
Lac Operon
Milkshake Powerade
POWERade is a drink manufactured by The Coca-Cola Company.
Lac Operon
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Induction by negative or positive control
Negative regulation: end-product repression
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