Flashcards in Week 3 L1 - Prokaryote Gene Regulation Deck (31)
Basic outline of the central dogma?
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.
Prokaryotes vs Eukaryotes - two main differences?
* 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.
How are most prokaryotic genes organised?
* 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
What is a promoter?
The transcription initiation sequence (DNA) in front of RNA start site.
What is an operon?
A group of genes transcribed from the same promoter
– common in bacteria and viruses.
What is a repressor?
A molecule that blocks transcription from the promoter.
What is an activator?
A molecule that increases gene transcription of a gene or set of genes.
What is an operator?
The site (DNA) at which the repressor binds to block transcription.
What is an inducer?
A molecule that induces transcription from the promoter.
What is induction?
The synthesis of gene product(s) in response to an inducer.
General Features of an Operon
* 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.
What are constitutive operons?
* 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.
What are regulatory operons?
• 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.
What controls the transcription of regulatory operons?
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.
When Lactose is the sole C source for E. coli, the synthesis of which 3 enzymes is induced?
• 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.
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?
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.
What type of transcription does lacI undergo? What is the pathway of events that occur here?
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.
The lac operon is negatively controlled. What does this actually mean?
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.
What is IPTG (Isopropyl-beta-D-1-thiogalactopyranoside) and what does it do?
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.
What happened when scientists created lacI and lacO mutants to test the theory of their function?
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.
Another mutation in the lacI gene was the lacIs mutation. What did this do and what were the results?
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.
The lac operon is negatively regulated by what?
The lac repressor.
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.
Cyclic AMP and the Catabolite Activator Protein (CAP)
* 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.
What is the positive regulator molecule of the lac operon?
The cAMP-CAP complex.
Relationship between glucose and cAMP levels in E. Coli?
Inversely proportional. cAMP is synthesized from ATP in a reaction catalyzed by adenylate cyclase which is negatively impacted by glucose.
What are the two methods of regulation of the tryptophan operon?
* 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.
What does the attenuator do in regards to regulation?
• 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
How many regions are there in the leader mRNA of tryptophan?
Four regions, with complimentary sequences which allow the formation of secondary structures.