Week 3 L1 - Prokaryote Gene Regulation

Question 1

Basic outline of the central dogma?

Answer 1

DNA (via transcription) to RNA, then RNA (via translation) to protein. Not usually able to go from protein back to RNA, but in viruses, can go from RNA to DNA using reverse transcription.

Question 2

Prokaryotes vs Eukaryotes - two main differences?

Answer 2

• Compartmentalisation - for example, eukaryotes have a nucleus, and prokaryotes do not.
• Introns - prokaryotes don’t have introns so the RNA that’s synthesised can be used directly, whereas in eukaryotes it needs to be processed and transported to the cytoplasm first.

Question 3

How are most prokaryotic genes organised?

Answer 3

Into operons.
* Prokaryote genomes
– much smaller than eukaryotes’
– usually less genes
– arrangement of genes is much denser
* Two or more genes often grouped & expressed together
– e.g., E. coli has >4000 protein-coding genes and most are in such groups
* These gene groups or expression units are called operons
* An operon has one promoter controlling several genes

Question 4

What is a promoter?

Answer 4

The transcription initiation sequence (DNA) in front of RNA start site.

Question 5

What is an operon?

Answer 5

A group of genes transcribed from the same promoter

– common in bacteria and viruses.

Question 6

What is a repressor?

Answer 6

A molecule that blocks transcription from the promoter.

Question 7

What is an activator?

Answer 7

A molecule that increases gene transcription of a gene or set of genes.

Question 8

What is an operator?

Answer 8

The site (DNA) at which the repressor binds to block transcription.

Question 9

What is an inducer?

Answer 9

A molecule that induces transcription from the promoter.

Question 10

What is induction?

Answer 10

The synthesis of gene product(s) in response to an inducer.

Question 11

General Features of an Operon

Answer 11

• Promoter, operator, structural genes (1,2,3 etc)
• An operon consists of a group of genes but only one promoter and usually only one transcription terminator at the end.
• Forms a polygenic (polycistronic) mRNA - single transcript = multiple proteins.
• Some operons are controlled by a regulatory element called an operator.
  – In such cases there is also a regulatory gene involved
  – which may or may not be part of the operon.
• Can get constitutive operons and regulatory operons.

Question 12

What are constitutive operons?

Answer 12

• Some operons contain genes that are expressed constitutively, i.e. similar levels at all times under all conditions.
• Usually, they are the “house-keeping genes”
  • eg. those for cell wall proteins.
• Consists of a promoter, gene 1, gene 2, gene 3, terminator.
  *They are NOT REGULATED.

Question 13

What are regulatory operons?

Answer 13

• Contain genes that are switched on and off, depending on certain stimuli
• i.e., lactose operon: where gene expression occurs only after induction
* Consists of promoter, operator, gene 1, gene 2, gene 3, terminator.
* Transcription (mRNA synthesis) and translation (protein synthesis) occur only after induction.

Question 14

What controls the transcription of regulatory operons?

Answer 14

The regulatory proteins.
Transcription to occur:
• Positive regulation: an ACTIVATOR protein must bind to DNA on the activator binding site (before the promoter).
• Negative regulation: a REPRESSOR protein must be prevented from binding to DNA (repressor proteins bind at the operator, after the promoter.
• Many genes interact with both activator and repressor
• The binding regions on DNA are called cis-elements, because they are on the same piece of DNA, right next to each other.

Question 15

When Lactose is the sole C source for E. coli, the synthesis of which 3 enzymes is induced?

Answer 15

• beta-galactosidase (lacZ gene) - lactose hydrolysis. Breaks the lactose into glucose and galactose (hydration reaction), and every so often it instead converts lactose to allolactose (changes the beta-1,4 linkage to a beta-1,6 linkage). Allolactose was shown to be the lactose operon inducer, rather than the actual lactose.
• lactose permease (lacY gene) - lactose transport. It embeds in the cell membrane, allowing lactose to move freely from outside the cell to inside the cell. This occurs at a much greater rate than the passive transport that occurs otherwise.
• thiogalactosidase transacetylase (lacA gene) - cellular detoxification? Not completely understood.

Question 16

Adding lactose only to the medium of E.Coli induces beta-galactosidase activity. What happens when both lactose and glucose are added to the medium?

Answer 16

Glucose is the preferred energy source, so will be used first. Therefore, there will be a delay of the induction (production) of beta-galactosidase whilst the glucose is being used, and then there will be the spike in beta-galactosidase induction once there is only lactose left and available to use.

Question 17

What type of transcription does lacI undergo? What is the pathway of events that occur here?

Answer 17

Constitutive transcription –> lac repressor mRNA –> ribosomes / translation –> lac repressor proteins. The lac repressor proteins are being constantly produced, all the time (i.e. constitutive transcription). Lac repressor proteins oligomerise, forming a tetramer that binds to the lacO (operator) site and prevents transcription of mRNA, i.e. lacI blocks expression. of the entire operon. RNA polymerase cannot bind to the promoter when repressor is bound to the operator.

Question 18

The lac operon is negatively controlled. What does this actually mean?

Answer 18

The lac operon is inhibited by the lac repressor proteins that are produced constantly. However, in the presence of allolactose (the ‘inducer’), the allolactose binds to the repressor protein causing an allosteric shift in shape and preventing it from binding to and inhibiting the lac operon, which is an example of negative control. Therefore, the lac operon is ‘switched on’ in the presence of allolactose.

Question 19

What is IPTG (Isopropyl-beta-D-1-thiogalactopyranoside) and what does it do?

Answer 19

It is a synthetic inducer, that unlike allolactose, doesn’t get metabolised in the process, but the desired effect of switching the operon on is still achieved without the deterioration of the IPTG.

Question 20

What happened when scientists created lacI and lacO mutants to test the theory of their function?

Answer 20

The lacI mutant produced inactive repressor proteins, and the lacO mutant created a site that the repressor proteins were unable to bind to. In both cases, there was constitutive expression of the lacZ, the lacY and the lacA proteins, both in the presence of lactose and without it.

Question 21

Another mutation in the lacI gene was the lacIs mutation. What did this do and what were the results?

Answer 21

It formed a ‘super-repressor’, that was unable to even recognise allolactose (the inducer). Therefore it would bind to the operator (lacO) and render the operon uninducible. In both the presence and absence of lactose, there was no transcription of genes lacZ, lacY and lacA.

Question 22

The lac operon is negatively regulated by what?

Answer 22

The lac repressor.

Question 23

Catabolite repression

Answer 23

Even a little glucose, in the presence of lactose
will make lac expression very low, since glucose is used preferrentially (i.e. used first). Glucose also reduces the level of cAMP in the cell, which is also important in lac operon expression. So essentially, the expression of beta-galactosidase is repressed by glucose.

Question 24

Cyclic AMP and the Catabolite Activator Protein (CAP)

Answer 24

• cAMP binds to the catabolite activator protein (CAP, dimer) to form cAMP-CAP complex.
• CAP is a transcription factor but only active when in association with cAMP.
• It binds to the CAP site in lac operon, recruits RNA polymerase to the promoter, increases rate of transcription. Example of positive control of lac operon.

Question 25

What is the positive regulator molecule of the lac operon?

Answer 25

The cAMP-CAP complex.

Question 26

Relationship between glucose and cAMP levels in E. Coli?

Answer 26

Inversely proportional. cAMP is synthesized from ATP in a reaction catalyzed by adenylate cyclase which is negatively impacted by glucose.

Question 27

What are the two methods of regulation of the tryptophan operon?

Answer 27

• Repression - when tryptophan is abundant, it binds to the repressor and activates it, allowing it to bind to the operator section of the promoter region and blocking transcription.
• Attenuation - the process to terminate transcription to form a short mRNA.

Question 28

What does the attenuator do in regards to regulation?

Answer 28

• Region of RNA sequence that forms secondary structures (hairpin loops in the RNA) that govern the level of transcription of attenuated operons.
• Part of the leader RNA

Question 29

How many regions are there in the leader mRNA of tryptophan?

Answer 29

Four regions, with complimentary sequences which allow the formation of secondary structures.

Question 30

Attenuator pairings of tryptophan RNA leader region.

Answer 30

Pairing of:

• Regions 1 and 2 – Transcription pause signal (ribosome loaded and translation follows).
• Regions 2 and 3 – Anti termination signal (allows transcription to continue).
• Regions 3 and 4 – Termination signal (stops further transcription).

Question 31

What happens when there is an abundance of trp and trp-tRNA in the cell? What if it is lacking?

Answer 31

• When there is an abundance, the ribosome reaches the double trp codons, adds the two trp-tRNA, and continues to zoom along to the end of region 2, which prevents region 2 from pairing with region 3. Instead, region 3 pairs with region 4, signalling termination, and the 5 trp genes that code for the enzymes that make more trp, do not get transcribed. This results in a ‘short’ transcript.
• If Trp is starved, ribosome stalls at the tandem Trp codons because Trp-tRNA is in short supply. Region 2 has chance to pair with 3, so the antiterminator formed. This prevents formation of the terminator (region 3 pairing with region 4). Transcription continues to trpEDCBA genes, i.e. a ‘long’ transcript.