week 1 - gene regulation in bacteria

Question 1

Gene regulation in bacteria

Answer 1

• regulation of lac
• simple bacterial regulation
• global (complex) regulation

Question 2

Regulation of lactose utilisation

Answer 2

• Simple system
  o Only two proteins required to use lactose as a carbon source (knew that he needed two genes to grow e coli):

Question 3

Regulation of lactose utilisation
what proteins needed

Answer 3

 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

Question 4

Regulation of lactose utilisation
when do e coli make these proteins

Answer 4

o E coli only makes these proteins when lactose is available in the media
 Therefore expression is regulated by lactose

Question 5

The lac operon
- - Genes at the lac locus:
structural genes

Answer 5

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)

Question 6

The lac operon
- - Genes at the lac locus:
regulatory genes

Answer 6

o LacI
 Regulatory region
 Mutations in i usually cause consitiuitive Lac+ phenotype
 E coli makes LacZ and LacY even in absence of lactose

Question 7

The lac operon
- - Genes at the lac locus:
How does LacI work?
experiment

Answer 7

• 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”

Question 8

The lac operon
- - Genes at the lac locus:
How does LacI work?
experiment 1

Answer 8

• 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)

Question 9

The lac operon
- - Genes at the lac locus:
How does LacI work?
experiment 2

Answer 9

• 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)

Question 10

The lac operon
- - Genes at the lac locus:
How does LacI work?
experiment 2
3 possibilities

Answer 10

• 3 possibilities
  1. lacI is segment of DNA that 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

Question 11

Identification of genes from sequence

Answer 11

• 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

Question 12

bacterial genes are transcribed:

Answer 12

• as individual genes
• in blocks of genes of related function
• both of the above

Question 13

bacterial genes are transcribed:
how are bacterial genes organised?

Answer 13

close together

Question 14

bacterial genes are transcribed:
e.g. when trp is avaliable

Answer 14

o Shuts off production
o To save energy
o (wont synthesise if don’t need it)

Question 15

Genetic regulation: central dogma

Answer 15

split into transcriptional and post transcriptional (after RNA)

Question 16

Genetic regulation: central dogma
increased proteins levels could be caused by:

Answer 16

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

Question 17

Genetic regulation: central dogma
regulation is said to be

Answer 17

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)

Question 18

Genes are operons can be regulated:

Answer 18

• 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

Question 19

Lactose regulation of the lac operon
summary -> see notes for images

Answer 19

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

Question 20

Lactose regulation of the lac operon
What happens when we mutate different components of the lac operon

Answer 20

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

Question 21

The trp operon – regulation by the Trp repressor
compared to lac?

Answer 21

• Trp repressor
  o Works differently from lac
  o Want to make trp if there isnt any in environment

Question 22

The trp operon
What happens when we mutate different components

Answer 22

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

Question 23

What might be the biological significance of the difference in regulation between the trp and lac operons

Answer 23

• 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

lac operon – Catabolic pathway (Breaks down lactose)

trp operon – Anabolic pathway (Synthesizes tryptophan)

Question 24

what else can genes be regulated by

Answer 24

activator proteins

Question 25

regulation by proteins if repressor is mRNA made if bound to operator

Answer 25

no

Question 26

regulation by proteins if repressor is mRNA made if not bound to operator

Answer 26

yes

Question 27

regulation by proteins if activator is mRNA made if not bound to operator

Answer 27

NO

Question 28

regulation by proteins if activator is mRNA made if bound to operator

Answer 28

yes

Question 29

if E coli growth with both glucose and lactose - Why don’t we see b-galactosidase synthesis until glucose is used up

Answer 29

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.

Question 30

The lac operon is regulated by a second protein: CRP (aka CAP)

Answer 30

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

Question 31

When is the lac operon transcribed

Answer 31

lactose low, glucose high: very low lactose low, glucose low: very low lactose high, glucose low: high lactose high, glucose high: low

Question 32

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

Answer 32

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

Question 33

summary - Cells regulate their final levels of proteins at many levels

Answer 33

o Transcriptional o Post-transcriptional o Post translational

Question 34

summary - Many genes/operons are regulated transcriptionally by

Answer 34

REPRESSPRS that binds to DNA and decrease RNA polymerase activity

Question 35

summary - Genetic approaches can be used to

Answer 35

dissect the mechanisms of unknown regulatory systems

Question 36

REPORTER GENES in vivo

Answer 36

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

Question 37

REPORTER GENES in vitro

Answer 37

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

Question 38

REPORTER GENES in vivo approaches - genetics requires a..

Answer 38

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

Question 39

REPORTER GENES in vivo approaches reporter genes - what?

Answer 39

- 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

Question 40

REPORTER GENES in vivo approaches what makes a good reporter?

Answer 40

- 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)

Question 41

REPORTER GENES in vivo approaches examples of reporter genes B-galactosidase

Answer 41

genes: lacZ reports on: gene expression, localisation

Question 42

REPORTER GENES in vivo approaches examples of reporter genes GFP

Answer 42

genes: gfp reports on: gene expression, localisation, redox environment

Question 43

REPORTER GENES in vivo approaches examples of reporter genes bacterial lucierase

Answer 43

gene: luxAB reports on: gene expression

Question 44

REPORTER GENES in vivo approaches examples of reporter genes alkaline phosphatase (phoA)

Answer 44

gene: phoA reports on: localisation, redox environment, secretion

Question 45

REPORTER GENES in vivo approaches examples of reporter genes firey luciferase

Answer 45

gene: luciferase reports on: protein folding

Question 46

REPORTER GENES in vivo approaches examples of reporter genes - fluorescence and gene expression

Answer 46

- For gene expression: Always get a bit of fluorescence in e. coli o So need a large enough difference to distinguish

Question 47

REPORTER GENES in vivo approaches transcriptional fusions

Answer 47

- 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

Question 48

REPORTER GENES in vivo approaches typical promoter probe vector Plasmid vectors have at least two features:

Answer 48

- 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

Question 49

REPORTER GENES in vivo approaches typical promoter probe vector multiple cloning site

Answer 49

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

Question 50

REPORTER GENES in vivo approaches typical promoter probe vector reporter gene

Answer 50

o Encodes a protein with an easily assayable activity

Question 51

REPORTER GENES in vivo approaches typical promoter probe vector how does it work

Answer 51

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

Question 52

REPORTER GENES in vivo approaches typical promoter probe vector example of use in studying gene regulation

Answer 52

- 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

Question 53

REPORTER GENES in vivo approaches typical promoter probe vector example of use in studying gene regulation GadB

Answer 53

- 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

Question 54

REPORTER GENES in vivo approaches what is another way to use transcriptional fusions

Answer 54

promoter analysis

Question 55

REPORTER GENES in vivo approaches Use of transcriptional fusions to determine properties of promoters in detail:

Answer 55

- 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

Question 56

REPORTER GENES in vivo approaches reporter fusion versatile tool

Answer 56

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

Question 57

REPORTER GENES best understanding

Answer 57

It is best to have complete understanding of in vivo and in vitro approaches:

Question 58

REPORTER GENES combination of in vivo and in vitro

Answer 58

- 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

Question 59

What might be the biological significance of the difference in regulation between the trp and lac operons lac operon

Answer 59

Catabolic pathway (Breaks down lactose) Purpose: To metabolize lactose (a sugar) when it is available. Default State: OFF when lactose is absent. Turned ON: When lactose is present and glucose is low. Reason: It would be wasteful to produce enzymes for lactose metabolism when lactose isn’t available. Cells prioritize glucose (simpler to metabolize); only switch to lactose when needed.

Question 60

What might be the biological significance of the difference in regulation between the trp and lac operons trp operon

Answer 60

Anabolic pathway (Synthesizes tryptophan) Purpose: To synthesize tryptophan (an essential amino acid) when it’s not available. Default State: ON when tryptophan is absent. Turned OFF: When tryptophan is present. Reason: It is energetically expensive to make tryptophan. If tryptophan is already available in the environment, there’s no need for the cell to produce it.

Question 61

What might be the biological significance of the difference in regulation between the trp and lac operons catabolic pathways

Answer 61

Turn ON only when substrate is available.

Question 62

What might be the biological significance of the difference in regulation between the trp and lac operons anabolic pathways

Answer 62

Turn OFF when end product (tryptophan) is available.

Question 63

REPORTER GENES in vivo approaches typical promoter probe vector how does the GadB gene gene respond over time to a drop in pH?

Answer 63

Initial Response: When E. coli encounters an acidic environment (low pH), it activates the gad (glutamate decarboxylase) operon, which includes the gadB gene. The acidic pH acts as a signal that triggers the GadE protein, which is a key regulator of this response. Over Time: Initially, GadE activates the gadB gene. This activation allows the cell to produce GadB (glutamate decarboxylase), which decarboxylates glutamate to γ-aminobutyric acid (GABA), a process that consumes protons and helps raise the internal pH of the cell. This mechanism is crucial for maintaining cellular homeostasis in low pH environments. Long-term Adaptation: Over time, the level of GadB enzyme activity helps the cell adapt to the acidic environment. However, as the pH stabilizes, the regulation of gadB may be reduced, and the system returns to a more neutral state once the acidic stress is alleviated.

Question 64

REPORTER GENES in vivo approaches typical promoter probe vector Does GadE Activate or Repress gadB?:

Answer 64

GadE Activates gadB: GadE is a transcriptional activator of the gad operon. When the pH drops and acid resistance is needed, GadE binds to the promoter regions of the gad genes, including gadB, and stimulates their transcription. This leads to the production of GadB, which helps the cell counteract the effects of low pH. Therefore, GadE is essential for the activation of gadB under acidic conditions.