Heather knight Flashcards
Explain the process of elongation
RNA polymerase unwinds the DNA as it goes and makes a bubble.
- Free ribonucleotide triphosphate bind to form the new chain.
- The polymerase makes sure they are the correct matches using its proof reading function.
- not as good as DNA polymerase but then it doesn’t need to be
What is the function of RNA polymerase in elongation?
It catalyses the formation of phosphodiester bonds that link nucleotides together in a chain.
- same as in DNA
The 5’ end, ends with a nucleotide in which the triphosphate group has not joined in a linkage with to make a phosphodiester bond.
The 3’ OH. Group attaches the 5’ phosphate of new nucleotide triphosphate to allow the chain to grow
What does the inverted repeat sequence of the terminator sequence in termination result in?
Mean that the synthesised RNA molecule will pair to itself and form a hairpin structure.
What does the formation of the hairpin structure cause?
May help to pull the transcript away from the RNA polymerase’s active site. In bacteria there are 2 types of termination signal.
What happens after coding sequence of a gene is transcribed into mRNA?
Transcription must stop. The RNA polymerase will only release the growing chain when it encounters a termination signal.
What structure do the termination signals in bacteria form?
Stem and loop, secondary structure through H bonding between Gs and Cs
What characterises intrinsic terminators?
A C-G rich stem followed by a run of As in the template strand.
How does the structure of the intrinsic termination sequence aid it’s function?
Hairpin structure is very stable.
- this makes it more difficult for DNA RNA base pairing to continue in the in the Hubble and will disrupt the progress of the RNA polymerase along the DNA.
- in addition when the run of A’s in the terminator sequence is encountered and transcribed by the polymerase, these results in AU pairs with only 2 H bonds compared with 3 for each of the GC pairs in the stem loop.
- in conclusion the stem loop structure is preferred to the ‘bubble’ do it is hard for the hybrid DNA RNA to stay attached and the RNA chain is released
What is a possible role for the flap structure?
RNA hairpin may be in contact with the flap structure.
Movement of the flap may contribute towards the breakage of the RNA-DNA hybrid and the ultimate termination chain.
What are Rho-dependant terminators?
These require a protein called Rho for termination of transcription. Rho attaches itself to the transcript as it moves along the RNA toward the polymerase.
How do Rho dependant terminators stop transcription?
Rho catches up and uses its activity as a helicase enzyme to break the base pairs between the DNA and RNA, stopping transcription.
Why is it important that the amount of a gene being transcribed varies hugely?
Not all proteins are required in the same amounts or at the same time. Switching genes in or off when needed saved energy
Explain the process of transcription initiation step 2
Once mRNA has begun to be synthesised, the sigma factor is released from the holoenzyme and the process of RNA polymerase moving down the chain starts in a more efficient manner.
What are the 3 types of transcriptional control mechanisms used by bacteria?
- Operons: coordinated control of gene groups
- Alternative sigma factors: decides which genes are transcribed
- Regulating transcription termination: decides whether addition genes are transcribed
What are 2 types of operons and give an example of each?
Catabolic (lac operon)
Biosynthetic (Trp operon)
Can be controlled by positive or negative mechanisms
What are a group of genes with related function called?
An operon
What is an operon?
A group of genes controlled by a shared promotor.
Genes on one operon are transcribed together to form a polycistronic mRNA what is this?
mRNA consisting of a continuous transcript that represents several different genes.
What is positive control in relation to onerous?
+ve control mean the DNA binding protein binds and switches transcription ON. -ve control is the opposite.
What is catabolism in terms of opera singer?
Is breaking down molecules to make something that is needed. Catabolic onerous control the expression of enzymes used in sugar metabolism and utilisation.
- Lactose Oberon is catabolic because they will only express enzymes when they are required I.e. If glucose is not avalible as a food source.
What is the structure of Lactose, why can’t E.coli break it down?
It is a disaccharide made up of 2 monosaccharide sugars joined together. These two sugars are glucose and galactose. E.coli can use the monosaccharides but cannot use the disaccharides.
Glucose and galactose are joined together by a 1,4 linkage. Bacteria cannot utilise this sugar so need to split it into its two component parts using an enzyme Called beta galactosidase.
What are the 3 steps called that enable lactose to be utilised?
- Detection- to see if lactose is avalible outside the cell
- Import- to import it into the cell
- Cleavage- to cleave into 2 sugars it can use.
Regulation of the lactose Oberon is ‘coordinate’ what does this mean?
All the enzymes are regulated in an identical fashion. The three genes lacZ/Y/A constitute an Oberon. They are structural genes and are transcribed from a single promoter into polycistronic mRNA. V rare in eukaryotes.
What are structural genes with regards to opereons?
They are concealed with the business of the Oberon, they code for enzymes.
This is in contrast to regulatory genes where they are concerned with the control of the Oberon.
What is special about LacI?
It has its own promoter and terminator this showed that it is a regulatory gene because when they disrupted its function by muting it all of the other three genes were expressed all the time.
- when it’s not disrupted we see control of the expression of LacZ T and A under different conditions.
What is the function of LacZ?
Codes for the enzyme beta-galactosidase which cleave lactose into galactose and glucose. Glucose is used immediately, when used up galactose induces another different Oberon to then be used up).
What does LacY code for?
Lactose per ease which transports lactose into the bacteria as it cannot simply diffuse across the plasma membrane.
What does LacA code for?
Transactylase.
What is the function of LacI?
Encodes a repressor (tetramer)
What is LacO?
Is next to lacZ and is the operator sequence.
When lactose is is not present, what happens?
LacI gene is transcribed and produces mRNA that is translated into a protein.
- 4 copies of this protein aggregate to form a tetramer.
- the tetramer is a repressor which binds to the operator region in the lac operon’s promoter and stops transcription occurring.
How does steric hindracne prevent the expression of the operon?
In the absence of lactose the repressor protein is transcribed (it always is by default). And acting as a tetramer, binds to the operator site. This interferes with the binding of RNA polymerase to the promoter by steric hindrance.
- the binding sites of RNA polymerase at the promoter and lac repressor at the operator overlap, hence the binding sites of RNA polymerase at the promoter and lac repressor at the operator overlap.
When there is no lactose what happens at the operator site?
The repressor protein binds preventing transcription of the Lac operon.
How does lactose induce expression?
By inactivating the lac repressor. Inducer converts lac repressor into an inactive form that cannot bind. RNA pol binds at promoter and transcribes the operon.
What effect does permease have in the lac operon?
Imports a few molecules of lactose and this is enough to start the next step. The inducer binds to the repressor and causes it to undergo a conformational change so that it can no longer recognise and bind to the base sequence of the operator. Thus RNA polymerase can now get to work to express the operon.
Why is there always some permease and beta-galactosidase present?
Everything in biology is in equilibrium- nothin is absolute.
Repressor (unbound) + operator DNA Repressor (bound)* Operator DNA
Lactose is not really the inducer. What is? and how is it produced?
beta-galactosidase can produce some side products and rearrange lactose rather than cleave it.
- one of the side products is allows toes which is beta-galactose 1-6-glucose.
- It is allolactose that is the inducer, this is present whenever lactose is.
What happens to the repressor molecules when lots of lactose is present?
All repressor molecules have bound allolactose.
What happens when the repressor is not bound to the promoter?
RNA polymerase DOES have access to the promoter because the repressor has been deactivated due to the binding of allolactose. The 3 structural genes are transcribed and later translated into enzymes.
How did Jacob and Monod gain the proof for the lactose operon?
E.coli mutant strains that didn’t normally respond to lactose.
- the mutations were in the genes that encode the regulatory system of the lac operon.
- they isolated two types: lacI- and LacOc- mutations
What is characteristic of a LacI mutant?
They are constitutive
- lacZ is always expressed
- LacI- is a mutant version of the LacI gene that encodes a mutant form of repressor that cannot bind to LacO, so expression is always on. > RNA polymerase can always bind to the promoter.
- so the constitutive effect is the constitutive expression of LacZ
Look at heather 1/4
Pages referring to mutants and their effects read though.
How is the catabolism of glucose able to repress the lactose operon? (I.e. When glucose and lactose is present, because the repressor-allolactose complex will still form allowing RNA polymerase to transcribe)
The process takes place involving a small molecule called cyclic AMP.
What does AMP catalyse?
The cyclisation of ATP into cyclic AMP.
What effect does high glucose have on cAMP?
High glucose means low cAMP
What did E.coli cya mutants show us?
They have defects in the enzyme adenyl cyclase and are unable to make cAMP. They are therefore unable to activate any of the ‘sugar operons’.
When treated with cAMP the mutant strains immediately developed operon inducability.
Therefore cAMP must be required for inducability.
What is the name given to the glucose phosphotransferase system?
The phosphoenol pyruvate.
How does the glucose phosphotransferase system work?
Protein IIA-Glc (is specific to glucose). When glucose is present the transport past these enzymes it becomes dephosphorylatesd. Preventing the conversion of ATP to cAMP. As cAMP is required for transcription of the operon, nothing is transcribed.
How does cAMP aid transcription?
It binds to a receptor called CAP (catabolise gene activator protein).
cAMP allows CAP to bind to the CAP site.
If glucose levels are high what happens to cAMP levels, what position is CAP and what is the result with regards to transcription?
Glucose high = cAMP low + CAP detached + no transcription of lacZ
When glucose is not present and the IAG1c is phosphorylated what events follow?
This form of the protein stimulates adenyl cyclase to produce cAMP and this allows the CAP protein to attach to the lac gene promoter and transcription can occur.
What happenes if both glucose and lactose are available to the cell?
CAP is detached and the lac operon is not transcribed even though the presence of lactose means that the repressor has Ben inactivated. The effect of glucose takes precedence over everything.
What is the function of CAP?
CAP binds to DNA in the promoter.
- the CAP binding site in the promoter consists of palindromic sequences.
What effect does CAP have on DNA?
CAP bends the DNA to force it apparat.
- the CAP protein is required to break the strong bond. It is fairly rigid so effort is needed to get it to bend.
What are the structural genes of the trp operon?
It has 5 structural genes, trp E/D/C/B/A that encode the trp repressor.
How do the needs of the trp operon differ to lac operon?
They are opposite in function.
E.g. If tryptophan is present in the growth media the bacteria do NOT need to synthesise it.
What effect does tryptophan have on the repressor.
It represses the expression of the operon.
- the repressor by itself is inactive, it is the aporepressor.
- it must complex with tryptophan itself the corepressor before it is active (the halo repressor).
What effect does the haloporepressor have on the trp operator?
It binds to the trp operator (trpO) and inhibits transcription by steric hindrance.
What is negative control with respect to regulation?
What is induction, what is repression?
It is a repressor.
Induction = inactivating the repressor
Repression = letting the repressor work
What is positive control with respect to regulation?
- what does induction do?
- what does repression do?
It acts as an activator
- induction = means letting the activator work. Activator is present and allowed to work.
- repression = stopping the activator from working. Lacks activator or a non-functional version that cannot work
What control mechanisms do the lac repressor and trp use?
Negative control.
If the lacI and trpR genes are deleted or inactive we get constitutive expression of the operons.
This means that in the absence of a regulatory control the operons are transcribed and translated.
To regulate them we have to switch them off.
If an operon is regulated by positive control what happens if gene for a regualtory protein is inactivated or deleted?
The operon will never be expressed.
In the absence of the activator protein in positive regulation what happens?
There is no expression at all. Even a low conc of activator is enough to switch on transcription.
What form of regulation of operon is only known to occur in eukaryotes and how does it work?
Positive control- repression.
A positive agent binds to the activator and makes it inactive.
What is special about the Arabinose operon?
It uses both positive and negative control.
TL-Arabinose is another such which if necessacery can be catabolised if no glucose is avalible.
Arabinose operon is regulated by both positive and negative control and it has 2 regulator DNA sequences: araO and araI.
How is the Arabinose operon controlled?
The AraC protein acts as a repressor. It binds to the operator araO and to araI. In doing so causes the DNA to from a loop that results in repression of transcription of the three structural genes (araBAD)
- in the presence of Arabinose itself, Arabinose binds to the AraC protein and converts it to an activator protein. This now binds to at the araI site pans switches on transcription. Like in lac operon the switching on also requires cAMP-CAP.
What are alternative sigma factors as a method of transcription regulation?
They decide which genes are transcribed.
Which sigma factor is used to switch on the expression of most ‘housekeeping genes’?
The sigma70
What does the sigma70 recognise?
-35 box.
What effect having would a different sigma factor have when used by RNA polymerase?
It would recognise a different protein. Used in situations for switching on and off different groups of genes in bacteria.
What effect does adding a different sigma factor to the core RNA polymerase enzyme have?
It causes a different holoenzyme to be made. These different versions having different sigma factors- will have different recognition capabilities.
What induces the synthesis of alternative sigma factors?
Particular conditions, including conditions (such as heat shock) that cause protein miss-folding.
Under heat shock what sigma factor is produced, and what are the differences to sigma 70?
Sigma32. It has different recognition properties and doesn’t recognise the consensus promoter sequences instead it recognises different ones. These are found in the promoter of heat shock genes (genes that are required to be switched on specifically to deal with heat shock)
What promoters to the sigma32 recognise?
Induced by unfolded proteins in the cytoplasm.
Genes encoding chaperones that re-fold the unfolded proteins and protease systems leading to the degradation of unfolded proteins in the cytoplasm.
What promoters to do the sigma factor sigma54 recognise?
Genes for nitrogen metabolism and other functions.
Regulating transcription termination: what is antitermination?
It allows terminator signals to be ignored.
How does antitermination work?
- RNA polymerase moves along the promoter and when it reaches antiterminatior protein it picks it up and brings it with it. RNA pol ignores a termination signal and carries on synthesising transcript until a second signal is reached.
- this is one way of allowing further coding regions to be transcribed.
- Antitermination is an important part of the infection stratagem of phage lambda.
What controls nearly all amino acid bio synthetic operons?
Attenuation.
How can the formation of stem and loop structures alter the termination of gene transcription?
You can either have stem-loops 1 and 3, then you make a terminator or you can have stem-loop 2 in which case you do not get a terminator.
What is the name given to the ‘short gene’ that overlaps stem and loops 1 and part of 2 coding for a 14 aa polypeptide?
A small open reading frame (ORF)
What formation of stem and loops always stops transcription?
Unless prevented by a mechanism the stem and loops will dorm in the order in which they are transcribed, so stem and loop 1 forms first, which precludes stem and loop 2 thereby allowing stem and loop 3 to form.
What happens to transcription/translation with high Trp?
Peptide is translated. Ribosome dissociates.
The end of ORF is before the second stem and loop structure is completely transcribed.
- so once the ribosome dissociates the first stem and loop strucutre will form, leading eventually it the formation of the 3rd stem and loop and resulting in transcription termination.
What happens to transcription/translation when there is low Trp?
Under reduced tryptophan concentrations ribosomes will stall over the trp codons.
This prevents stem and loop 1 from forming. Eventually stem and loop 2 will be transcribed and form.
This precludes the 3rd stem and loop from formin and thus prevents transcription termination.
RNA polymerase will now progress into the operon allowin it’s expression and the synthesis of more tryptophan.
In 5 bullet point summarise attenuation:
- Terminator mRNA BEFORE the biosynthetic genes.
- It’s formation is prevented if a ribosome stall in a crucial position.
- The ribosome is translating a short ORF into a protein that need the AA that is to be synthesised.
- So … If AA is present, ribosome won’t stall and terminator will be formed.
- Some operon only use attenuation and don’t use repressor sources at all.
What are bacteriapahges?
Virus’s that infect bacteria.
What is the structure of the bacteriophage lambda?
Has a head and tail structure.
Double stranded linear DNA chromosome of about 48 kilo base pairs.
What are the 4 stages of a hypothetical simple virus?
- Virus’s DNA (that encodes a single type of viral protein coat) enters the cell
- The virus genome is replicated producing multiple copies.
- The genome copies are translated and transcribed to produce the viral protein coat.
- The viral genomes can the assemble spontaneously with the coat protein to form new virus particles which escape from the cell by lysing it.
How do bacteriophage co-ordinate the expression of genes to facilitate transition through these stages of the developmental program?
The first genes to be expressed (usually straight after infection) produce a protein that will allow the switching on of genes that are required later.
These are often referred to as late or early genes.
Early gene expression > protein coded by one early gene > switches on expression of the late genes > late gene expression
What is the choice that the bacteriophage lambda must make? Why must it make this decision?
Lysogeny or lytic pathway.
- if it is going to make lots of new phage particles it is only sensible to make them I’d there are lost of new bacterial hosts for them to invade.
- therefore if there are not many unoccupied hosts or little nourishment for the virus then it is sensible for them to stay put (the lysogeny pathway).
What is the definition of a temperate phage?
It can sit and wait to decide what pathway to choose.
(Phage lambda is an example of this).
A host with a lysogen is immune to further infection.
What are virulent phages?
Always opt straight for the aggressive lytic option.
What are bacteriapahges?
Virus’s that infect bacteria.
What is the structure of the bacteriophage lambda?
Has a head and tail structure.
Double stranded linear DNA chromosome of about 48 kilo base pairs.
What are the 4 stages of a hypothetical simple virus?
- Virus’s DNA (that encodes a single type of viral protein coat) enters the cell
- The virus genome is replicated producing multiple copies.
- The genome copies are translated and transcribed to produce the viral protein coat.
- The viral genomes can the assemble spontaneously with the coat protein to form new virus particles which escape from the cell by lysing it.
How do bacteriophage co-ordinate the expression of genes to facilitate transition through these stages of the developmental program?
The first genes to be expressed (usually straight after infection) produce a protein that will allow the switching on of genes that are required later.
These are often referred to as late or early genes.
Early gene expression > protein coded by one early gene > switches on expression of the late genes > late gene expression
What is the choice that the bacteriophage lambda must make? Why must it make this decision?
Lysogeny or lytic pathway.
- if it is going to make lots of new phage particles it is only sensible to make them I’d there are lost of new bacterial hosts for them to invade.
- therefore if there are not many unoccupied hosts or little nourishment for the virus then it is sensible for them to stay put (the lysogeny pathway).
What is the definition of a temperate phage?
It can sit and wait to decide what pathway to choose.
(Phage lambda is an example of this).
A host with a lysogen is immune to further infection.
What are virulent phages?
Always opt straight for the aggressive lytic option.
