Lac operon

Question 1

How is gene expression regulated?

Answer 1

Genes can be turned on and off like switches

Question 2

Prokaryotic gene expression

Answer 2

Highly efficient genetic mechanism that turns genes on and off, depending on the metabolic needs of the cell for that particular gene product.

Regulation is at the level of gene transcription

Question 3

Gene have different levels of expression

Answer 3

The E. coli genome encodes over 4000 proteins.

• Some proteins are present in very low number (e.g. 5 to 10) per bacterial cell.
• While other proteins are present in great numbers (e.g. 100,000) per bacterial cell.
• Hence, E. coli regulates the synthesis of gene products (proteins).

Question 4

Bacterial operons

Answer 4

Genes that encode the enzymes of a metabolic pathway are usually clustered together on the chromosome in a functional unit called operon

Question 5

List 7 characteristics of operons

Answer 5

• > An operon is a group of genes that are transcribed at the same time.
• > An operon usually consists of a few structural genes, a promoter and an operator region.
• > A regulatory DNA sequence is part of the operon.
• > The genes are transcribed as a single mRNA molecule called polycistronic mRNA.
• > The polycistronic mRNA is translated into a few separate proteins or enzymes.
• > These proteins or enzymes usually control an important biochemical process.
• > Operons are only found in prokaryotes

Question 6

Operons can be under inducible or repressible regulation

Answer 6

Transcription can be controlled by a repressor protein, which when bound to the operator site of the DNA.

It blocks the movement of RNA polymerase from the promoter to the structural genes.

Question 7

Inducible and repressible regulation work on the same principle

Answer 7

-> If the repressor is able to bind to the operator, the genes are turned off.

-> If the repressor is inactivated and unable to bind operator, the genes are
expressed.

Question 8

Inducible operon (lac operon)

Answer 8

• > The repressor binds to the operator and prevents the structural genes from being transcribed.
• > When the inducer is available, it binds to repressor, which then undergoes conformational change and detaches from the operator.
• > When repressor is away, RNA polymerase attaches to promoter and genes are transcribed

Question 9

Repressible operon (trp operon)

Answer 9

• > The repressor, by itself, is unable to bind to the operator and the structural genes are actively transcribed.
• > When the substrate is available, it acts as a co-repressor by binding to the inactive repressor.
• > Repressor undergoes conformational change, allowing it to attach to the operator, so RNA polymerase cannot transcribe genes.

Question 10

What is the lac operon

Answer 10

The lac operon codes for a cluster of genes that

regulates the production of enzymes needed to degrade lactose in bacterial cells.

Question 11

What are the 4 components of lac operon

Answer 11

• Promoter and Operator regions
• 3 structural genes (which code for enzymes) in the order Z, Y and A
• All 3 lacZYA genes are transcribed by RNA polymerase as a single polycistronic mRNA
• The 3 lacZYA genes are coordinately regulated since a single polycistronic mRNA codes for all three enzymes

Question 12

LacZ gene

Answer 12

• > Encodes for the enzyme b-galactosidase
• > The b-galactosidase cleaves the disaccharide lactose into the monosaccharides glucose and galactose
• > This conversion is essential if lactose is to serve as the primary energy source in glycolysis

Question 13

LacY gene

Answer 13

• > Encodes for the enzyme galactoside permease

- > Permease transport lactose into the cell

Question 14

LacA gene

Answer 14

-> Encodes for the enzyme galactoside transacetylase

It transfers an acetyl group from acetyl-CoA to galactosides, glucosides and lactosides

Question 15

Operator region

Answer 15

• > 27 base-pairs long DNA sequence
• > Located immediately before the lacZ structural gene
• > Regulates mRNA transcription of lacZYA genes but does not produce a gene product (protein)

Question 16

Promoter region

Answer 16

• > Contains two separate binding sites:
• one for RNA polymerase
• one for CAP-cAMP complex

-> RNA polymerase and CAP-cAMP complex need to bind to the promoter in order to initiate mRNA transcription

Question 17

lacI gene

Answer 17

• > Located close to the lacZYA genes (but are not part of the structural gene cluster)
• > Has its own promoter
• > Codes for a 360 amino acid Lac repressor protein
• > Lac repressor protein regulates the transcription of the lacZYA genes by binding to the operator region
• > Lac repressor is allosteric (i.e. the repressor reversibly interacts with another molecule, causing a conformational change in the shape of the repressor)

Question 18

What happens when lactose is present

Answer 18

• > If lactose is present, the concentration of enzymes responsible for lactose metabolism increases rapidly.
• > So, the enzymes are inducible, and lactose is the inducer.

Question 19

What happen when lactose is absent

Answer 19

-> If lactose is absent, then these enzymes are not produced.

Question 20

Why no lac-ZYA encoded enzymes are produced if lactose is absent?

Answer 20

• > The repressor protein is produced from the lacI gene.
• > The repressor protein binds to the DNA sequence of the operator region.
• > The repressor protein inhibits the transcription of the lacZYA genes by the RNA polymerase

Question 21

Why lac-ZYA encoded enzymes are not produced if lactose is present

Answer 21

• > The repressor protein is produced from the lacI gene.
• > The repressor protein binds to the available lactose which causes a conformational change in the shape of repressor protein.
• > The repressor protein is incapable of binding to the operator DNA.

Question 22

Catabolite repression

Answer 22

Catabolite repression is the mechanism that represses the lac operon when glucose is present.

Bacteria prefer to use glucose when there is both lactose and glucose available because lactose needs to be hydrolyzed.

Lac operon will not be activated until the cell metabolizes all the glucose present.

It is only activated until glucose supply is used up.

Question 23

Co-operative binding

Answer 23

By RNA-polymerase and cAMP-CAP

RNA polymerase cannot bind to the promoter efficiently on its own and requires the assistance of a “helper protein complex”.

Neither both have strong affinity to bind to lac promoter.

But when they are placed together on lac promoter, a tightly bound complex is formed.

Question 24

Helper protein complex consists of ?

Answer 24

1. catabolite activator protein (CAP)

2. cyclic adenosine monophosphate (cAMP)

Question 25

Summary of cooperative binding

Answer 25

Both RNA polymerase and CAP-cAMP complex are required to bind to their respective sites within the promoter in order for efficient transcription of lacZYA genes.

Question 26

How does glucose affect catabolite repression?

Answer 26

- > CAP binding to CAP binding site is dependent on cAMP (i.e. for CAP to bind to CAP-binding site, CAP must be linked to cAMP). - > The level of cAMP is itself dependent on an enzyme adenyl cyclase, which catalyzes the conversion of ATP to cAMP:

Question 27

Relationship of the concentration of glucose to the concentration of cAMP in the cell

Answer 27

Low glucose concentration cause high cAMP concentration in the cell. High glucose concentration cause low cAMP concentration in the cell.

Question 28

Summary of glucose regulating catabolite repression

Answer 28

- > High levels of glucose inhibit the enzyme activity of adenyl cyclase, so adenyl cyclase cannot convert ATP to cAMP. - > This causes a sharp fall in cAMP levels in the cell. - > Thus, there is no cAMP present to bind to CAP. - > CAP (without cAMP) cannot bind to the CAP binding site in the promoter and consequently, RNA polymerase cannot bind to RNA binding site in the promoter in the absence of the CAP-cAMP complex. • There is no mRNA transcription of the lacZYA genes when glucose is present in the media.

Question 29

One line summary again

Answer 29

RNA polymerase cannot bind to the promoter efficiently in the presence of lactose and glucose, due to the absence of the CAP-cAMP complex. Hence, transcription of lacZYA genes does not occur.

Question 30

4 possible situation when glucose and lactose is present or absent

Answer 30

When both are present, E.coli does not produce beta galactosidase When both are absent, E.coli does not produce beta galactosidase When glucose is present and lactose is absent, E.coli does not produce beta galactosidase When lactose is present and glucose is absent, E.coli produces beta-galactosidase.

Question 31

LacI- mutant

Answer 31

Repressor protein is mutated and cannot bind to operator region lacZYA structural genes are always turned on, regardless of whether lactose is present or absent The RNA polymerase binds at the promoter site, initiating transcription even though lactose is absent

Question 32

Oc mutant

Answer 32

Nucleotide sequence of operator DNA is altered and will not bind to a normal repressor molecule The lacZYA structural genes are always turned on (constitutive expression), regardless of the whether lactose is present or absent. RNA polymerase bind to the promoter, imitating transcription, whether or not lactose is present.

Question 33

LacZ- or lacY- mutant

Answer 33

If there is a mutation in the lacZ gene or lacY gene, then these mutants fail to produce active b-galactosidase or permease respectively, and are unable to use lactose as an energy source

Question 34

Combinations of mutations

Answer 34

refer to final slide