2) Control of Transcription

Question 1

Why is it important that the amount a gene is transcribed and translated be varied?

Answer 1

Not all proteins are required in the same amounts or at the same time

Switching transcription of genes on and off when needed SAVES ENERGY

E.coli: Only needs to transcribe genes needed to make proteins to use the food substrates

Question 2

How do prokaryotes control the amount of transcription?

Answer 2

1) Cell can control which genes are transcribed (where in the DNA transcription starts and stops)
2) Can control the rate and amount of transcription

Question 3

What are the 3 different types of control mechanisms used by bacteria? (Names Only)

Answer 3

1) Operons: Coordinate control of gene groups
2) Alternative sigma factors: Decides which genes are transcribed
3) Regulating transcription termination: Decides whether additional genes are transcribed

Question 4

What are operons?

What are the 2 types?

Answer 4

Genes which have related function in bacteria are grouped together and are regulated in a coordinate fashion= Operon

Controlled by a shared promoter

Regulatory proteins bind to the DNA of the promoter and its operator to regulate expression

Example: 9 genes making the 9 enzymes which synthesise histidine= Operon

Can be controlled by positive or negative mechanisms

2 types:

1) Catabolic (e.g. lac operon)
2) Biosynthetic (e.g. Trp operon)

Question 5

What is the difference between positive or negative control?

Answer 5

The DNA-binding protein that binds the promoter can be a positive or negative regulator

Positive= Transcription is switched ON

Negative= Transcription is switched OFF

Question 6

What the difference between catabolic and biosynthetic operons?

Answer 6

Catabolic: When the operon is involved in breaking down substrates. Produce molecules they can use for extracting energy

Biosynthetic: Synthesising new molecules they require for life

Question 7

Where are catabolic operons used?

Answer 7

Control expression of enzymes used in sugar metabolism and utilisation

Question 8

What happens to catabolic operons when glucose is available?

Answer 8

Glucose= Preferred sugar
Do not needed to switch on expression of enzymes which break down other compounds and release sugars= Transcription= OFF, normally OFF

These operons (such as Lactose (lac) and arabinose (are) are ONLY switched on when their sugar is present

Question 9

What does the lactose operon do?

Answer 9

Lac gene: Codes for the enzyme B-galactosidase which breaks down the sugar lactose= Cleaves the beta 1,4 linkage

Lactose: Disaccharide made up of 2 monosaccharide sugars joined together= Glucose + Galactose= E.coli can only use the monosaccharides

Question 10

What are the different genes of the lac operon?

Answer 10

lacZ, lacY and lacA= Lac operon AND also lacI

Structural genes and are transcribed from a single promoter into polycistronic mRNA

Question 11

What is the difference between structural and regulatory genes?

Answer 11

Structural genes: Code of enzymes or others that actually DO the work

Regulatory genes: Control the operon

Question 12

What is the regulatory gene for the lac operon?

Answer 12

LacI= Gene that produces the regulatory protein. It has its own promoter and terminator

Product of lacI regulates expression of lacZYA

If its function is disrupted, all of the other 3 genes are expressed ALL the time

It encodes a REPRESSOR

Question 13

What does lacZ do?

Answer 13

Encodes the enzyme beta-galactosidase which is very stable

Proof experiments: Always monitor lacZ as it is the easiest to detect= Only need to monitor one of them
Able to measure beta galactosidase activity easily= turns blue on X-gal

Question 14

What does lacY do?

Answer 14

Encodes a permease= Transports lactose into the bacteria as it cannot diffuse through the plasma membrane by itself

Question 15

What does lacA do?

Answer 15

Encodes an enzyme transacetylase= Add acetyl groups to toxic sugars= Makes them harmless
(Although no one really knows its function…)

Question 16

How is the lac operon regulated when lactose is ABSENCE?

Answer 16

Also have: lacO= Next to lacZ, is the operator sequence= Site at which the repressor protein acts

1) lacI gene is transcribed= produces mRNA that gets translated to a protein
2) 4 copies of proteins aggregate together= Tetramer
3) IF THERE IS NO LACTOSE: tetramer can bind to the operator region (lacO)
4) Binding= Interferes with binding of RNA polymerase to the promoter by steric hinderance (gets in the way…)
5) Prevents the expression of operon= Transcription is switched OFF

Binding sites of RNA polymerase at the promotor and lac repressor OVERLAP= if protein binds= Gets in the way= RNA polymerase cannot bind

Question 17

How is the lac operon regulated when lactose is PRESENT?

Answer 17

Prevents the repression mechanism from working

1) Always have some permease in the plasma membrane= Imports lactose
2) Beta-galactosidase can rearrange lactose= Produces side product allolactose= Inducer
3) Allolactose binds to lac repressor protein= Conformational change= inactive form that cannot bind to operator
4) RNA polymerase can bind to the promoter and transcribes the operon= Structural genes are transcribed and translated into enzymes

Allolactose is present whenever lactose is present

Question 18

What does the lacI- mutant do?

Answer 18

lacI- mutant version of lacI gene= Produces version of the lac repressor protein which CANNOT BIND to the operator

Result: Expression of the 3 structural genes of lac operon are ALWAYS switched on= Transcription ON

lacZ gene is constitutively expressed= Their effect is to cause constitutive expression of lacZ and the other genes

Question 19

How did Jacob and Monod discover the Operon model?

Answer 19

Took 2 strains of chromosomes:
F’= Extra inserted genes delivered on a small plasmid
+= Normal, effective wild type version
-= Defective mutant
1) lacI- lacZ+ / F’ lacI lacZ-
Chromosome has mutant lacI, F’ plasmid adds normal lacI joined to mutant lacZ

2) Lac+ lacZ+/ F’lac- lacZ-
Chromosome has normal versions of both genes, F’ plasmid adds mutant version os both

Asked question: Would extra copies of genes (on F’ plasmid) alter behaviour of original strain

Question 20

What happened in strain 1?

Answer 20

Normal lacI gene on F’ plasmid makes normal version of repressor protein= Normal repression and induction
SHOWS: lac+ (wild type) is the DOMINANT as it can overcome the lac- mutation + the gene must encode a diffusible product that can move to the operator on the bacterial chromosome= MUST have the lac repressor protein

Strain 2: Also normal induction

Question 21

What does it mean by “lacI acts in TRANS with respect to the lac operon’?

Answer 21

The lacI gene does not need to be on the same piece of DNA as the lac operon as there is a diffusible product that can move to the DNA when required= Lactose repressor

Question 22

What do lacO^c mutations do?

Answer 22

Cis-dominant

LacO^c = cannot be recognised by a functional wild type repressor (lacI+)= Protein cannot bind= Operon is constitutively switched on

Question 23

How did their cis-dominant property be discovered by Jacob and Monod?

Answer 23

Again took 2 strains:
1) lacOc lacZ+ / F’lacO+ lacZ-
Mutant and normal wild type lacZ is on bacterial chromosome, F’ plasmid adds normal wild type lacO operator with mutant lacZ

RESULT: Still constitutive, adding the extra wild type lacO does not help

(ii) lacO+ lacZ+ / F’lacOc lacZ-
Normal wild types on bacterial chromosome, and mutants on F’ plasmid added
RESULT: Normal, the 2 mutations on the plasmid do not destroy normal functioning

SHOWED: When lacO gene is on the SAME chromosome as the functional lacZ, it is dominant= Cis-dominant
LacO cannot produce a product that can diffuse and affect genes somewhere else

Question 24

What does the lacI^s mutations do?

Answer 24

Super-repressed mutations and are un-inducible

RESULT: Make repressor insensitive to inducer= Mutant repressor will always be bound to the operator= Transcription is always switched OFF