week 1 - gene regulation in bacteria Flashcards
Gene regulation in bacteria
- regulation of lac
- simple bacterial regulation
- global (complex) regulation
Regulation of lactose utilisation
- Simple system
o Only two proteins required to use lactose as a carbon source (knew that he needed two genes to grow e coli):
Regulation of lactose utilisation
what proteins needed
B-galactosidase
* Enzyme that cleaves lactose (a disaccharide consisting of a galactose with a B linkage to glucose) into glucose and galactose
* However will act on any b-galactoside
Lactose permease
* Transporter protein for lactose
o Lets lactose into the cell where b-galactosidase can act on it
Regulation of lactose utilisation
when do e coli make these proteins
o E coli only makes these proteins when lactose is available in the media
Therefore expression is regulated by lactose
The lac operon
- - Genes at the lac locus:
structural genes
o lacZ
encodes G-galactosidase (sometimes called LacZ)
mutations in z cause Lac- phenotype (e coli cannot ferment lactose) and disrupt B-galactosidase activity
o LacY
Encodes lactose permease (sometimes called LacY)
Mutations in Y cause Lac- phenotype but does not disrupt B galactosidase activity
o lacA
encodes lactose acetylase (sometimes called lacA)
The lac operon
- - Genes at the lac locus:
regulatory genes
o LacI
Regulatory region
Mutations in i usually cause consitiuitive Lac+ phenotype
E coli makes LacZ and LacY even in absence of lactose
The lac operon
- - Genes at the lac locus:
How does LacI work?
experiment
- Transfer lac region from Hfr into F- recipient
a. Interrupt mating using waring blender/screen for recombinants
When did DNA transfer?
b. Measure LacZ activity
When is B-galactosidase expressed? - IPTG/TMG = indice expression of lacZYA, BUT not a substrate for B galactosidase
o “gratutitous inducer”
The lac operon
- - Genes at the lac locus:
How does LacI work?
experiment 1
- Hfr strain
o In presence of:
Streptomycin (kills donor)
IPTG (turns on lac) - Lag between transfer of genes and production of B galactosidase:
o Takes time to turn gene into a “gene product” - Lac + recombinants after 15 mins
o Lac is about 10 mins from origin of cell - B-galactosidase activity at about 20 mins
o Lag between genotype (gene entering cell) and phenotypes (expressing gene)
The lac operon
- - Genes at the lac locus:
How does LacI work?
experiment 2
- In the absence of inducer neither parent can make B-galactosidase
- However during mating recipient
1. Starts to make B-galactosidase;
THEN
2. Turns off synthesis of new B-galactosidase - PROBLEM: Can see lag before production of LacZ
o Why are we getting expression of b-galactosidase in the recipient cell? (burst before wanes off)
The lac operon
- - Genes at the lac locus:
How does LacI work?
experiment 2
3 possibilities
- 3 possibilities
1. lacI is segment of DNA tat senses lactose directly (cis-acting)
2. lacI makes a product that is required for synthesis of B-galactosidase
3. lacI makes a product that shuts off synthesis of B-galactosidase - interpretation
1. production of new gene products takes time
2. both z and I make products
3. production of I (lacI) binds DNA and “represses” expression of z
4. lactose (and IPTG/TMG) inhibit LacI
there was a 3rd experiment that confirmed this
Identification of genes from sequence
- translate the DNA in all six reading frames
o three forward (top strand), three reverse (bottom strand) - looking for “open reading frames” (ORFs)
o stretches of bases that start with an ATG and end with a stop codon (TAA, TGA, TAG) - note however not all ORFs will encode proteins
bacterial genes are transcribed:
- as individual genes
- in blocks of genes of related function
- both of the above
bacterial genes are transcribed:
how are bacterial genes organised?
close together
bacterial genes are transcribed:
e.g. when trp is avaliable
o Shuts off production
o To save energy
o (wont synthesise if don’t need it)
Genetic regulation: central dogma
split into transcriptional and post transcriptional (after RNA)
Genetic regulation: central dogma
increased proteins levels could be caused by:
increase in amount of mRNA made
(post-transcriptional)
increased stability of mRNA once made
increase translatability of mRNA
- regulate ribosome to bind start codon better
increase stability of protein once made
Genetic regulation: central dogma
regulation is said to be
transcriptional: if it affects amount of mRNA made
post-transcriptional: if it affects the stability of the mRNA or the translatability of the mRNA
post-translational: if it affects the stability (or some other important property) of the protein)
Genes are operons can be regulated:
- Negatively
o Transcription inhibited
o E.g. by binding of a repressor protein - Positively
o Transcriptionally stimulated
o E.g. by binding of an activator protein - Both of the above
Lactose regulation of the lac operon
summary -> see notes for images
LacI binds the operator and turns off transcription of lacZYA
- When LacI binds to operator
- Prevents RNA polymerase from accessing the promoter
- So no transcription
Lactose binds to LacI and prevents LacI from binding to O, which turns on transcription of lacZYA
- Lactose binds to LacI
- No longer binds to repressor
- Synthesis
- So works in presence of lactose
Lactose regulation of the lac operon
What happens when we mutate different components of the lac operon
state of lac operon in absence of any lactose or glucose
WT : OFF
deletion of repressor: ON
deletion of lac promoter: OFF
deletion of lac operator: ON
lac repressor unable to bind operator: ON
lac repressor unable to bind lactose/PTG: OFF
The trp operon – regulation by the Trp repressor
compared to lac?
- Trp repressor
o Works differently from lac
o Want to make trp if there isnt any in environment
The trp operon
What happens when we mutate different components
state of trp operon in absence of tryptophan
WT: ON
deletion of a repressor: ON
deletion of trp promoter: OFF
deletion of trp operator: ON
trp repressor unable to bind operator: ON
trp repressor unable to bind lactose/PTG: ON
What might be the biological significance of the difference in regulation between the trp and lac operons
- Why is the lac operon OFF when lactose is absent but the trp operon is ON when tryptophan is absent?
o Need to consider anabolic and catabolic reactions
what else can genes be regulated by
activator proteins
regulation by proteins
if repressor if mRNA made if bound to operator
no
regulation by proteins
if repressor if mRNA made if not bound to operator
yes
regulation by proteins
if activator if mRNA made if not bound to operator
NO
regulation by proteins
if activator if mRNA made if bound to operator
yes
if E coli growth with both glucose and lactose
- Why don’t we see b-galactosidase synthesis until glucose is used up
The lac operon is regulated by a second protein: CRP (aka CAP)
- Catabolite activator protein (CAP) acts as a glucose sensor. It activates transcription of the operon, but only when glucose levels are low. CAP senses glucose indirectly, through the “hunger signal” molecule cAMP.
The lac operon is regulated by a second protein: CRP (aka CAP)
e.g. by itself by Plac promoter only weakly recruits RNA polymerase
CRP recruits RNA to Plac promoter
- CRP required cyclic AMP (cAMP) which is made by adenylate cyclase to be functional
o cAMP binds CRP
o CRP then binds to CRP operator
o Glucose negatively regulates synthesis of CAMP
Glucose inhibits adenylate cyclase activity
The adenylate cyclase activity is required for CRP to activate transcription
When is the lac operon transcribed
lactose low, glucose high: very low
lactose low, glucose low: very low
lactose high, glucose low: high
lactose high, glucose high: low
How could you determine if a given operon or gene is under positive or negative control?
- Think about effects of deleting the genes for the repressor or activator proteins
see table
go through:
WT
Deletion of activator (CRP)
Deletion of promoter
Deletion of operator
Activator unable to bind operator
Activator unable to bind inducer
summary
- Cells regulate their final levels of proteins at many levels
o Transcriptional
o Post-transcriptional
o Post translational
summary
- Many genes/operons are regulated transcriptionally by
REPRESSPRS that binds to DNA and decrease RNA polymerase activity
summary
- Genetic approaches can be used to
dissect the mechanisms of unknown regulatory systems
REPORTER GENES
in vivo
In vivo means measuring something that has been made by the living cell (e.g. mRNA, protein)
- This may be while the cell is still living or after the mRNA/protein ad been extracted
- Advantages: comes closer to what a cell actually does
- Disadvantages: cells are complex, often mechanistic details are lost
REPORTER GENES
in vitro
In vitro means reproducing some aspects involved in the expression of mRNA or protein in the test tube, using purified components (e.g. DNA, RNA polymerase, mRNA precursors)
- Advantages: can control components making it possible to investigate fine details of mechanism
- Disadvantages: may not be what happens in the cell/may be oversimplified
REPORTER GENES
in vivo approaches
- genetics requires a..
phenotype
o Genetic is interaction between genotype and phenotype
o To know whether a process is on or off need a phenotype
- But many biological processes do not have (useful) phenotypes
REPORTER GENES
in vivo approaches
reporter genes - what?
- A “reporter gene” is a genetic tool to express a phenotype that “reports” on a particular biological process
o E.g. gene expression localisation, stability, protein, RNA production, redox environment, protein folding
REPORTER GENES
in vivo approaches
what makes a good reporter?
- Should have a simple assay to easily measure activity (e.g. B-galactosidase activity, fluorescence, luminescence)
o In other words a quantifiable phenotype - Changes in biological processes (e.g. increased gene expression) should correlate to changes in activity (increased B-gal activity)
- Low or zero background activity that can be attributed to other processes (i.e. controls)
REPORTER GENES
in vivo approaches
examples of reporter genes
B-galactosidase
genes: lacZ
reports on: gene expression, localisation
REPORTER GENES
in vivo approaches
examples of reporter genes
GFP
genes: gfp
reports on: gene expression, localisation, redox environment
REPORTER GENES
in vivo approaches
examples of reporter genes
bacterial lucierase
gene: luxAB
reports on: gene expression
REPORTER GENES
in vivo approaches
examples of reporter genes
alkaline phosphatase (phoA)
gene: phoA
reports on: localisation, redox environment, secretion
REPORTER GENES
in vivo approaches
examples of reporter genes
firey luciferase
gene: luciferase
reports on: protein folding
REPORTER GENES
in vivo approaches
examples of reporter genes
- fluorescence and gene expression
- For gene expression: Always get a bit of fluorescence in e. coli
o So need a large enough difference to distinguish
REPORTER GENES
in vivo approaches
transcriptional fusions
- Fuse the promoter of interest to a gene whose product is easily assayed
- This is a transcriptional fusion
o Most useful for this group - Also called promoter probe fusions
- But the reporter downstream of the promoter of the regulatory gene we are studying
REPORTER GENES
in vivo approaches
typical promoter probe vector
Plasmid vectors have at least two features:
- An origin of replication
o Ensures plasmid replicates and determines its copy number - A selectable marker (most common is antibiotic resistance)
o E.g. AmpR, TetR, KanR
o Enables selection of cells containing plasmid following DNA transformation
REPORTER GENES
in vivo approaches
typical promoter probe vector
multiple cloning site
o Several different restriction enzymes sites in the DNA to facilitate cloning of fragments containing promoters
o Typical multiple cloning sites:
Several different restriction sites (cute with restriction enzyme)
Cut only once in the plasmid
Promoter fragments can be inserted using one site only or any combination of two sites
REPORTER GENES
in vivo approaches
typical promoter probe vector
reporter gene
o Encodes a protein with an easily assayable activity
REPORTER GENES
in vivo approaches
typical promoter probe vector
how does it work
o Joining the promoter under study to a gene with a product which is easy to assay
o In this case lacZ that encodes beta-galatosidase
- Now when we measure the beta-galactosidase activity we are actually measuring the level of expression from the promoter of interest
REPORTER GENES
in vivo approaches
typical promoter probe vector
example of use in studying gene regulation
- The gadB gene is required for acid resistance in e. coli
- It is regulated by the GadE protein
o How does the gadB gene respond over time to a drop in pH
o Does GaE activate or repress gadB - Activity of GadB is hard to measure
REPORTER GENES
in vivo approaches
typical promoter probe vector
example of use in studying gene regulation
GadB
- Make a transcription fusion between the gadB promoter and lux reporter genes
- Measure Lux activity (LIGHT) after addition of acif in a WT and in a gadE deletion mutatnt
o Light production tells us something about how gadE is being expressed - Bacteria containing this plasmid will emit light under conditional where the gadB promoter is active
- Time course of acid induction of gadB in a WT (grey) or gadER deletion mutant (black) background
- Is gadBB activated or repressed by GadE in the presence of acid
o Wanted to know if gadE was an activator or repressor
o Can see an increase is light production (activity) after adding acid to cells (more gadB)
o gadE must be an activator
Because if got rid of gadE and it was a repressor would expect it to be produced all the time
But this is not what happens
REPORTER GENES
in vivo approaches
what is another way to use transcriptional fusions
promoter analysis
REPORTER GENES
in vivo approaches
Use of transcriptional fusions to determine properties of promoters in detail:
- Measure expression in different mutant backgrounds
o To determine role of proteins that regulate the promoter
o Can include activity in presence/absence of regulator protein expressed from a plasmid - Measure expression over time
o To determine response to specific conditions and changes in conditions - Measure expression after mutagenesis of promoter DNA
o To determine role of specific sequences in the promoter
REPORTER GENES
in vivo approaches
reporter fusion versatile tool
Reporter fusion are a very versatile tool
- Transgenic Drosophila expressing LacZ in specific places at different stages of development
- Transgenic frog with tissue specific expression of GFP in eye lens cells
REPORTER GENES
best understanding
It is best to have complete understanding of in vivo and in vitro approaches:
REPORTER GENES
combination of in vivo and in vitro
- For example might have a mutated operator (e.g. by a single base change) that binds repressor less tightly than the WT operator
o In vivo: expect to see higher level of expression of operon under repressing conditions
o In vitro: purified repressor protein should bind to purified operator DNA more tightly than it binds to purified mutated operator DNA - They should give consistent results
o These kinds of experiments can allow us to do things like map the operator very precisely
Down to the individual bases of DNA recognised by the repressor
