Module Three Flashcards
what has selection led to?
the evolution of efficient systems
what is enzyme induction?
bacteria only produce enzymes required for growth on a particular substrate in the presence of an inducer
what is the function of the promoter in a gene?
binds RNA polymerase and initiates transcription
what is the function of the operator?
binds regulatory proteins to alter transcription
what is negative regulation?
gene expression is turned off by a regulatory protein termed a repressor
what is positive regulation?
gene expression is turned on by a regulatory protein termed an activator
when is a gene expressed?
when the activator is active and the repressor in inactive
when is a gene not expressed?
when the activator in inactive and the repressor is active
what controls the lac operon?
a repressor protein
what happens to the lac operon when the activator is not present?
the repressor protein binds and inhibits the binding of RNA pol II
when is the lac operon transcribed?
only in the presence of lactose
who won the Nobel prize for discoveries concerning genetic control of enzyme and virus synthesis?
Francois Jacob
Jacques Monod
André Lwoff
what was the hypothesis surrounding inducers and gene activation?
that inducers that led to increased product activated a pre-existing enzyme
what was the beta-galactosidase, inducer experiment?
an inducer was added and the amount of beta-galactosidase increased
when the inducer was removed the synthesis stopped
what is the inducer of the lac operon?
Lactose is an inducer (allolactose)
what else can act as an inducer of the lac operon?
IPTG is a related molecule that is not a substrate and can be used as an inducer of the lac operon
what are the genes contained within the lac operon?
lacZ
lacY
lacA
what did the PaJaMo Experiment find?
The I+ allele could repress b-gal in the presence of I-
The I+ (wild-type) was dominant over I-
which lacI is dominant?
lacl+ is trans-dominant over lacl-
what encodes for a diffusible repressor of the lac gene?
lacl
what happens in the presence of lacl+ and lacl_?
lacl+ encodes for the repressor which can bind both genes
gene is not expressed
what happens in the presence of lacl^s?
lacl^s mutants do not express lacZ, lacY or lacA
lacl^s is dominant over both lacl+ and lacl-
the repressor cannot bind the inducer
how do O^c mutants affect the binding of repressors?
the repressor cannot bind to the mutant operator (O^c)
the gene is expressed even in the presence of an inducer
what is a virus?
an infectious agent that must grow or reproduce inside a host cell
what is a virus composed of?
nucleic acid, genetic material and protein coat (some also contain lipids in their coats)
what is a bacteriophage?
Viruses that infect bacterial cells
what is phage lambda?
an phage that infects E.coli
what are the features of phage lambda?
Double stranded DNA genome (dsDNA)
Long flexible tail, icosahedral head
what does the lifecycle of phage lambda depend on?
depends on the state of the host bacterium
what is the lytic lifecycle?
results in the lysis of the bacterial cell upon release of progeny phage
what is the lysogenic lifecycle?
results in the stable carriage of the phage (prophage) within the host cell (lysogen)
what is a virulent phage?
A phage that is only able to undergo replication via the lytic cycle
what is a temperate phage?
A phage that can replicate either in the lytic or lysogenic cycles
what is a lysogen?
a host cell that is harbouring a prophage during lysogeny
what is a prophage?
the latent for of a temperate phage that remains within the lysogen
what are the important events in the lambda phage lifecycle?
infection
decision (lytic or lysogenic)
maintenance
induction or ‘switch’
what are cos sites?
cohesive end sites
what is the structure of the genome in a phage head?
the genome in linear
when does the genome of the phage circularise?
following injection
what are the steps in phage assembly and release?
- head and tail proteins are synthesised
- DNA is packaged into heads
- tails are added
- the host is lysed releasing new phage
what are the steps in phage integration and maintenance?
- lambda intergrates
- prophage is stably maintained
- prophage passed to daughter cells
what are the sites where integration occurs between the phage and bacterial chromosome?
attP (attachment on Phage)
attB (attachment on Bacteria)
what is the enzyme that integrates the phage into the bacterial chromosome?
Integrate
when is it a good time to make more phages?
High growth of bacteria, plenty of food/energy
when is it a good idea to integrate into the genome?
If the bacterial cell in unlikely to have the resources to produce many more phages
when is it a good time to leave the lysogenic cycle and enter the lytic cycle?
if the host cell is damaged and likely to die
what is the Cro protein?
Expressed early in infection and the major player in establishing lytic growth
what is the promoter that produces N protein?
PL
what is the promoter that produces Cro protein?
PR
what is N protein?
an anti-terminator protein that enables transcription past 2 terminators resulting in early gene expression
what are the phage genes involved in DNA replication (lytic growth)?
O and P
what are the phage genes involved in Recombination (integration)?
int and xis
when are the proteins for the lytic and lysogenic pathways produced?
proteins for both pathways are produced early before the phage has decided which cycle to enter
what is the nut site?
N utilization site
What is cro?
a DNA-binding protein that represses transcription
-cro promotes the lytic cycle
what is CII?
CII is a DNA-binding protein that activates transcription
-promotes the lysogenic cycle
what is CI?
is a DNA-binding protein that can activate or repress transcription
- Cl activates it’s own expression
- Represses genes required for lytic cycle
- maintain lysogeny
what protein promotes the lysogenic pathway?
Cll
what protein promotes the lytic pathway?
Cro
what is the deciding protein for which lifecyle the phage will enter?
Cll
what is the roll of Cll in deciding which lifecycle that phage will enter?
Host protease degrade Cll
Healthy cells produce high levels of protease
In actively growing cells, Cll gets degraded
when does the cell enter the lytic cycle?
in actively growing cells Cll is degraded and so there is lots of Cro protein and this promotes the lytic cycle
what is Q?
Q is an anti-terminator than enables expression of late lytic genes (head, tail and lysis genes)
when does a phage enter the lysogenic lifecyle?
starved cells produce less protease and so the Cll is not degraded
This promotes entry into the lysogenic cycle
What does Cll activate in the phage?
int and Cl repressor
what does int activation result in?
intergration of lambda into host chromosome
what does Cl repressor do?
activates its own expression and binds to OL and OR to repress all other phage genes
how is lysogeny maintained?
by Cl
how does Cl protein maintain the prophage?
Cl is a repressor of all phage genes but an activator of itself
Keeps the phage genome ‘silent’ in bacterial chromosome until induction
what promotes the lytic cycle?
Cro represses cl
Q anti-terminates lytic genes
what promotes the lysogenic lifecycle?
Cll activates cl and int
what types of plaques does lambda form and why?
Lambda forms turbid plaques due to lysogens being immune to further infections
what protein maintains lysogeny?
Cl
how does Cl maintain lysogeny?
Cl is a repressor of all phage genes but an activator of itself
keeps phage genome ‘silent’ in the bacterial chromosome
what are the basic components of the switch?
Regulatory proteins Cl (repressor) and Cro
Operator sites on the phage DNA and their promoters
RNA polymerase (essential for transcription
what are the two positions of the switch?
- Lysogeny: Cl on and Cro off (maintenance)
2. Lytic: Cl off and Cro on (induction)
what are the three operator sites for Cl and Cro?
OR1, OR2, OR3
what are the two promoters for RNAP?
PRM
PR
-do not overlap
What is RNAP?
RNA polymerase
transcribes DNA into RNA and is provided by the bacterial host
where does RNAP bind in the switch?
to either PR or PRM but never to both
PR activity with RNAP does not need a regulatory protein
PRM needs Cl as an activator
what happens when RNAP is bound to PRM?
Cl is activated
Lysogenic cycle is maintained
what happens when RNAP is bound to PR?
Cro is activated
The lytic cycle is induced
what are the features of the operators?
17bp long
similar to each other but Cl and Cro can distinguish between them due to different affinities
how do Cl dimers bind to DNA?
using their N-terminal domain
where do Cl dimers bind to DNA?
to one OR along one side of the DNA helix
what is negative control with Cl?
Cl at OR2 turns off cro gene by preventing RNAP from binding to cro promoter (exclusion)
what is positive control with Cl?
Cl at OR2 helps RNAP bind and begin transcription of cl gene
what are the two functions of cl?
negative control
positive control
what are the two factors that contribute to Cl binding?
- intrinsic affinity
- Cooperativity
when is the affinity for Cl increased?
When Cl is bound at OR1 it increases the affinity at OR2
What is the affinity for Cl at OR3?
weak and no cooperativity
How does the cooperative binding of Cl work?
Dimers of Cl ‘lean’ towards each other
Why does cooperative binding not occur at OR3?
the Cl C-terminal domains are unavailable
what is the binding of Cl in the maintenance of lysogeny?
Cl is bound at OR1 and OR2
This inhibits cro and maintain expression of cl
This state is very stable and inherited by daughter cells after cell division
what is induction?
- prophage excises from the host chromosome
2. Lytic growth
what can cause induction?
UV light
how does UV cause induction?
the operator sites are cleared of Cl by activated RecA
where do cro dimers bind?
to each OR site in the absence of Cl
Cro binds on one side of the DNA helix
What is the structure of Cro?
cro has a single domain
Two monomers or cro from dimers
almost all cro in the cell is dimeric
how does cro bind to the DNA?
cro binds to each operator site independently
when is possible Cl expression stoped?
when Cro is bound to OR3
