Transcription/Translation Flashcards
Requirements for transcription
- RNA polymerase
- Addition to 3’ end
- No Primer
- Promotor on DNA
Sigma factor
- RNA polymerase needs the sigma factor in order to recognize the promotor region. The sigma factor is a binding protein that will recognize the correct sequence and bind at start site of promotor
- Promotor will assemble and orientate RNA polymerase
Binding at negative 35 region
The tighter that RNA polymerase and Sigma factor bind the more robust transcription will be
Transcription terminators
specific sites that stop transcription in which it will form a stem loop with uracil and disengage
rRNA
are cotranscribed and then processed to form final rRNA
Control of gene expression
- transcriptional control-NO mRNA synthesis
- translational control-No enzyme synthesis
- post-translational control-Enzyme is modified to active or inactive
Post translational control
Feedback inhibition-final product inhibits first enzyme of pathway to control anabolic pathways to tweak amount of products being produced
Allosterism
Is a non-covalent interaction in which an inhibitor binds the allosteric site reversibly and changes the configuration so that is can no longer function. By increasing the concentration of end product you increase the activity
Covalent modifications
Changes the activity of the enzyme but can only be reversed if modified by second enzyme
Example: adenylation
Regulation at transcriptional level
- repression
- induction
- positive control
- global control
- stringent response
- quorum sensing
- attenuation
- signal transduction
Similar metabolic pathways are linked and have same promotor to yield a polycistronic message
Repression
- A way in which to control the amount of enzyme by decreasing the amount of mRNA encoding for enzyme in operon
- Arginine is lacking in the media and then is added and enzymes repress mRNA involved in arginine synthesis so growth is no interrupted
Induction
- A way in which to control the amount of enzyme by increasing the amount of mRNA encoding for enzyme in operon
- B-galactidase is induced when lactose is present in order to utilize it nutrients
Control of Transcription
At the promotor region certain enzymes have to bind to DNA and will occur because of protein interaction with specific domains and motifs of DNA
Helix turn helix
Protein domain that allows for binding of proteins to DNA which allows for control of gene expression at transcriptional level
1. Stabilizing helix, recognition helix and turn for flexibility
Zinc fingers
As zinc finger binds to DNA the zinc ion binds to a protein which holds elements by binding Histidine and cytosine in the middle
Repression of operon
- Operon-has regulatory and coding region
- Repressor will bind to operator region and will need a corepressor (arginine) in order to bind
- Repressor has allosteric site that when the corepressor binds allow for the change in conformation so that it can bind to operator.
- Corepressor is end product of anabolic pathway
- RNA polymerase bind to the promotor region and is unable to transcribe if repressor is bound
Induction of operon
- When lactose is unavailable then the repressor can bind to the operator and prevent transcription
- When lactose is present, then it binds to the repressor and changes its active site so that it can no longer bind to operator
Positive Control
- Activator proteins bind to activator binding site on RNA polymerase to enhance transcription
- Can be allosterically modified by inducers to promote transcription
- Inducers promote binding of activator protein to activator binding site to stimulate transcription
- Binding site is upstream from promotor
- ACTIVATOR PROTEINS CAN NOT BIND WITHOUT INDUCER
Regulon
Pathway that is spread throughout bacterial chromosome
Global regulatory mechanisms
LAC operon:
- Catabolic repression and efficient use of different carbon sources
- Many different genes are regulated at one time
LAC operon
- As glucose is starting to be depleted then the repression is lifted and B-galactidase is induced for catabolism of lactose
- Requires CAMP bound to CAP (CAMP/CAP) which can bind upstream of the promotor which needs to be bound in order for transcription to occur.
- As glucose is depleted CAMP and CAP are activated
Stringent Response
- Global control mechanism
- Triggered by AA starvation
- RelA produces alarmones (pGpp, and ppGpp) to monitor ribosome activity
- Alarmones are derived from Guanine nucleotide
- Balance protein production and protein requirement
- Alarmones stop rRNA and tRNA synthesis and Amino acid synthesis is activated in order to catch up before continuing transcription
Quorum Sensing
- Detect accumulation of released signals and change behavior when concentration exceeds threshold level
- Under Global control
- Sense population size by sharing specific small molecules and once a specific level is reached gene expression is triggered
- AHL (Acyl homoserine lactone) is a quorum sensing inducer
Action of AHL
- Cross membrane by diffusion and bound by LuxR regulatory proteins
- Binding occurs when Acyl goes into hydrophobic acyl binding site of LuxR
- Complex will bind to DNA promotor region and activates transcription of quorum sensing proteins
- Results in: light production, virulence, EPS secretion, motility, and Plasmid transfer
Attenuation
control transcription after initial RNA synthesis
Attenuation Characteristics
- Control gene expression by down regulation
- transcription and translation occurs simultaneously
- Used in amino acid biosynthetic pathways
- Transcription does not begin until level amino acid is low
Leader protein for attenuation
- Attenuation ribosomes translate leader mRNA to yield amino acid rich leader peptide that can sense sufficient amino acid in enviroment. It is enriched in amino acids of that pathway.
Excess tryptophan in attenuation
- Leader mRNA has code for excess amino acid
- Cells with excess amino acid has leader protein with amino acid
- In excess situation mRNA is fully translated and forms a stem loop to inhibit further transcription
Tryptophan Starvation in attenuation
- Leader mRNA has code for needed amino acid
- Cells that are starved will have this partially translated leader that lacks the amino acid so it will stall
- partially translated mRNA will form a different stem loop and inhibit further transcription until tryptophan levels are increased
Signal transduction
- Two component regulatory system
- Sensor kinase protein- autophosphorylation due to enviroment or can phosphorylate target cytoplasmic proteins (serine, threonine, tyrosine)
- Response regulatory protein- activity depends on phosphorylation
Sensor kinase proteins
transfer phosphate to response regulatory protein which is a DNA binding protein that acts as a repressor in the phosphorylated form
Phosphatase
- Dephosphorylates
2. Functions as a sensor when kinase is activated
Mutation
- heritable change in DNA that can lead to change in phenotype
- spontaneous or induced due to changes in base sequence due to insertions or deletions
mutant
differs from its parental strain in phenotype
selectable mutation
give mutant a growth advantage under certain environmental conditions
His-
means that organism can not make own histidine
Mutation rate
10-7 or 10-11 base pairs (10 million to 100 billion times a gene is replicated and will acquire a mutation
Mutagen
Chemical, physical agents, or biological agents that increase mutation rate above spontaneous rate of mutation.They can alter DNA in many ways but is not a mutation unless inherited
Substitute for thymine
5-bromouracil and can cause faulty pairing with G
Substitute for adenine
2-aminopurine and can cause faulty pairing with C
Radiation on mutations
Ultraviolet light and ionizing radiation both cause massive mutations so much that bacteria can not survive
UV radiation forms
Pyrimidine dimers in one strand
Ionizing radiation form
free radicals in cells
SOS regulatory system
Activated due to DNA damage and initiates a number of repair processes that are error-prone and high fidelity
How SOS works
- SOS is a regulon
- RecA protein senses DNA damage and inactivates LexA which is a repressor
- Then activates UmuD and UmuC which are catalytic subunits of PoLV
- This is an error prone polymerase
Ames Test
Sensitive bacterial assay system for detecting chemical mutagens in environment, food, drugs, and cosmetics
How Ames test works:
Back mutation: organisms mutates into a new mutant that is able to reverse outcome of 1st mutation
Ames test using His-
- Place bacteria on medium that lacks histidine and no growth will occur
- when a cell treated with a mutagen is added to medium then a back mutation takes place and growth does occur.
Homologous recombination
Closely related DNA from 2 distinct genetic elements combine into a single unit
How homologous recombination works
- Donor DNA has a nearly homologous strand that is wants to share with recipient DNA
- Endonuclease nicks the donor strand and SSB proteins stabilize DNA
- RecA is then used to facilitate the exchange and allows strand invasion
- Develop a cross strand exchange
- Ligase seals everything up
Three means of gene transfer
- Transformation
- Transduction
- Conjugation
Transformation
- Donor bacterium releases naked or free DNA when it is dying (stressed environment)
- Recipient bacteria must uptake it and remains alive by cell surface proteins at cell wall binding free DNA
- Must have competence to take up free DNA because it allows binding of DNA
Used to prove that DNA was the genetic material.
Live R cells and heat killed S cells still killed the animal because transformation in the R cells because of acquiring DNA from S cells
Transduction
- Depends on bacteriophages to transfer a gene from one bacteria to another bacteria
- Virus must inject chromosome disruption
- Donor-DNA with viruses is released
Conjugation
- Mediated by conjugative plasmids and circular DNA needed for bridge formation
Transformation needs:
- competence
- RecA-used to match homologies in DNA and resolve pieces
- SSB proteins -degrade 1 strand and incorporate 1 strand
- Restriction enzymes-degrade DNA coming into cytoplasm is it is not wanted
In the end: Single strand of DNA is incorporated into chromosome using homologous recombination and complementary strand is synthesized
Tranduction steps: Generalized and Specialized
Generalized
1. Virus attaches and injects its DNA into cytoplasm
2. Phage DNA overtakes the DNA machinery and host chromosomes are degraded into small pieces and die
3. Viral DNA with a capsule is released to infect new cells .
(lysis)
Specialized
- May have a random piece with host DNA and virus will then inject host DNA to be incorporated into chromosome (homologous recombination)-efficiency is low (1 in 10,000)
- DNA is carried in Capsid of bacteriaphage and if Donor DNA replaces all of viral genome in bacteriophage then the virus will be defective because it doesn’t have all the genetic information for its own replication
Specialized Transduction
- Viral DNA incorporated in host of Donor
- It is then excised from donor host chromosome
- IF adjacent host genes are excised with viral DNA then this virus is not defective
- transducing efficiency is high
Temperate virus
gene is able to replicate along with host and not cause cell death (lysogeny)
-Some can cause phenotypic changes in the bacteria they infect even without transducing bacterial genes
Provirus (prophage)
genome of a temperate virus replicating within host genome
Lysogen
bacterium with prophage
Lysogenic pathway
steps after virus infection leading to replication of viral genome as a prophage
Lytic pathway
steps after virus infection that leads to virus replication and destruction of the host cell (produce more viruses)
- phage injects DNA into host cell and replicated and genes are expressed where proteins then can assemble and release phage particles
- excursion of phage DNA from host to normal phage production without excising any DNA
Lysogenic state
- Viral genes confer new properties on cell
- Pathogens become toxic when bacteriophages inject DNA into it and became lysogenic because it had new properties of virulence
- phage DNA not replicated or transcribed and integrated into host genome
- Host replicates carrying phage genome
- Integrated Dna-prophage DNA
- When DNA is excised it enters the lytic cycle
Example: coryebacterium diphtheria or salmonella anatum
lambda phage (temperate phage)
virus genome gets incorporated into bacterial DNA at same spot each time
Normal:
1. Cell has Galactose gene is host DNA and you have a phage DNA in lysogenized cell
2. Phage DNA circularizes and detaches from host DNA
3. NO host DNA excursion and DNA replicated and phage synthesis is complete
4. Cells lysis and release normal phage
Rare:
1. when a portion of host DNA is exchanged for phage DNA and now phage is Galactose positive due to impercise excision
2. replicated and phage synthesis and cells release defective phage that can transduce galactose
Plasmids
- Small circular or linear DNA that carry unessential genes
2. Plasmids in a cell can NOT be closely related
Fertility plasmid
genetic map for E.coli
F plasmid
- controls conjugation and process of transferring DNA of donor plasmid to recipient cell
- Involves replication via a rolling circle mechanism
Plasmid reproduction
- autonomously
- control own replication( many copies of same gene but not closely related genes)
- direct conjugation
Plasmid conjugation
- transfer plasmid from one bacteria to another
Conjugation with plasmids give:
- plasmids have genes that give phenotype
- resistance to antibiotics
- production of toxins
- metabolize certain substrates such as pesticides and solvents
Increase antibiotics you will increase plasmid transfer and number
R plasmid
- Antibiotic resistance to as many as 5 at once
- By using conjugation is is rapidly diffused throughout bacterial population
Ex-Mursa staph aureus
Requirements for conjugation
- cell to cell contact
- bacterial mating
- plasmid or plasmid chromosome
- sex pilus of donor
- occur during replication
- only 1 strand transferred from donor
Conjugative plasmids
initiate gene transfer by altering cell surface to allow contact between F plasmid donor cell(F+) and F plasmid-less recipient (F-)
Plasmid genes code for:
production of sex pilus that initiates pair formation
Conjugation with rolling circle
- Cell pair is stabilized and F plasmid nicked in 1 strand
- Transfer gene from F+ to F- using a rolling circle mechanism while also replicating it in the F+ cell
- synthesis in complementary strand begins in recipient
- Complete transfer and synthesis and seperation
- It is now competent to serve as a donor (F+)
Hfr strains
Donor cell is mobilized for transfer to a recipient cell and F plasmid is integrated into host chromosome to produce Hfr.
- F plasmid inserts into host chromosome at a specific site (insertion site)
- replicates and transfers its self starting at middle of plasmid and drags entire host chromosome with it.
- Transfer takes up to 100 minutes for whole plasmid
- IF is interrupted then genes closest to F plasmid would be transferred so the new traits would be those close to F plasmid (would remain an F-)
Hfr stands for
high frequency of recombination
F plasmid (episome)
- integrated at a specific site
- specialized conjugated plasmid
- always mobilize host chromosome at same spot
- F plasmid prevents donor from being recipient
F’ plasmid
imprecise excision of host chromosome gives F’ that has a piece of DNA from host and if it doesn’t have any host chromosome DNA it is a F plasmid.
1. Include gene that affect phenotype on the F plasmid
Hfr goes to
- Has an integrated F plasmid so it becomes an F-
IS (insertion Sites)
these are specific sites that have regions of DNA homology between chromosome and F plasmid of DNA
Peeling off of plasmid to F- recipient
peels off and transfers closest host gene within minutes or 100 minutes for all genes to be transferred over
Temperate phage
Cause phenotypic changes in the bacteria they infect without transducing bacterial genes
