Reading...
Play button
Play button
Use LEFT and RIGHT arrow keys to navigate between flashcards;
Use UP and DOWN arrow keys to flip the card;
H to show hint;
A reads text to speech;
156 Cards in this Set
- Front
- Back
|
how big is bacteria genome
|
about 2x10^6 mbp
|
|
|
what is Genetic
|
is studying the organism as a whole and looking at the affect of the organism with mutation. How do you mutate a gene?
|
|
|
Prokaryote vs. eukaryote
|
-membrane bound organisms
-nucleus -prokaryotes = circular and linear, but they are specific to particular microbes |
|
|
Horizontal transfer
|
By plasmid
|
|
|
What’s in a bacteria cell chromosome
|
E.coli and salmonella… they have 1 circular
-vibrio….2 circular…….they have halfplod -borrelia (lyme)……. 17 chromosome…some are circular and some are linear -azotobacter (associated with roots, fix nitrogen) -Deinococcus (radiodurans)..can withstand radiation |
|
|
-Deinococcus (radiodurans)..can withstand radiation
|
Gama rays and x rays causes DNA breakage…we think that they have triplod or more so that when there’s a breakage. It hopes that the extra chromosome isn’t broken at the same spot…therefore, they are not mutated.
|
|
|
How is chromosome different than plasmids and viruses?
|
Chromosomes (both pieces) are pieces that are essential for any circumstances…for examples… rna polyermase, ribosome…etc…these are are Essential genes. Plasmids do not encode these essential genes. They are genes that are important for say growths…etc.
|
|
|
what is nucloid
|
As bacteria divides…the chromosome coil and the dense chromosome is called nucloid. They will need to make two to split to daughter cells.
Parent bacteria chromosome takes up about 1mm… the problems is how can we get 1mm into 2mm (e. coli size) Replication is one of the events where mutations arrive as well as the rate. |
|
|
types of coils
|
Dna supercoiling where the dna helix are coiled by enzymes.
Relax form takes up a lot of spaces. Where every part of every turn touches the surfaces AT rich regions at the bending of the supercoil |
|
|
important of negative coil
|
Negative coil goes left handed… and it creates a breathing zone. The dna that does it is called DNA gyzase… type II topoisomerase…type 2 because it cuts strands two at a times
|
|
|
essential DNA pol and important ones
|
Essential …Dna pol III = dnaE…major replication. 5-3 pol function and 3-5 exonucl function
Essential… Dna pol I .... can remove primers...one of them. 5-3 exonucl function….in the case where there’s lagging, it can get rid of the RNA of the next coming strands. Back up for removing rna. Cause there’s RNaseH… specific for strands with RNA and DNA Dna pol II... IV and V are important for repair |
|
|
3-5 exonucl.
|
error proofing enzyme. Therefore you have 1/1mil of mistaking = 1/1000 x 1/1000.
|
|
|
mutation.....DNA polymerase mistake.
missense mutation… |
always a point mutation…but it’s not exactly the same… point mutation means a one nucleotide changes. 1/10^6
|
|
|
Silent mutation
|
where there’s no phenotype differences. .. 1/3
|
|
|
Leaky mutation
|
have partial phenotypes or partial functions
|
|
|
mutation
failure to remove RNA primers |
a. will affect ½…. Have switching of A---U. where U are not remove
b. transition…replaces with an original group… purine to purine, and pyr- pyr c. transversion….purine to pyr and pyr to purine |
|
|
transversion….
|
purine to pyr and pyr to purine
|
|
|
transition…
|
replaces with an original group… purine to purine, and pyr- pyr
|
|
|
frameshift mutation
|
a. What causes shift? Plus One fs…the replication strain (new strain) slips out and comes back but it miss a line and comes back (like hair pin figure)…but a base is push out while the replication continue. Now the new strain has one more base pair.
b. Can have a plus two fs…when there’s a slipping off of the strain. What causes the shift? c. Minus one frame shift base…(the wild type strain or the template strain slipping) it loose it’s track but instead of the new strain slipping, the template slips. d. A minus two fs would be two base slipping out e. Consequences of the mutation f. Genes are transcript in groups called operons Usually, a plus one is a missense mutation. However shift mutation is worst due to the complete termination of the transcription |
|
|
deletions
|
when they are longer than 4 base. Many mechanisms
a. in chromosome… have repeated sequences called transposon…where ever they insert, they can cause mutation look at diagram in lecture notes 2. |
|
|
deletion and duplications
|
Cells with deletion and duplications gives cells a lot of freedom…however, if the deletion includes essential materials… it dies.
Other ppl think deletions are form by slippage when replicating Lagging strain….for some reason, the single strain binding protein doesn’t attach and the dna coil up…there’s a deletions of dna. if it doesn’t have pressure than it wouldn’t have it… with stress and the cells that mutated with the best genes…natural selection makes them the best. if it doesn’t have pressure than it wouldn’t have it because it takes a lot of energy to have more base pair (duplication) and the cells that mutate with the best genes…natural selection makes them the best. Cells does not delete or duplicate because they want to…but do you random selection in the face of stress. Cells that survive wins Cells with deletion and duplications gives cells a lot of freedom (also something that bacteria carries out when stress)…however, if the deletion includes essential materials… it dies. Other ppl think deletions are form by slippage when replicating |
|
|
reversion
|
Reversion frequency is 1/10^9
The reversion of a frame shift is about 1/10^5 – 1/10^6 Reversion to a deletion is zero because you cant get back the information that’s not there. With reversion…ppl can tell what type of mutation took place. if a bacteria cell have a leaky mutation (missense mutation/pt. mutation) say within the duplicated strain… the cells can revert the mutation to work. The frequency is 1/10^9 for reversion of a point mutation. This is hard and rare event. However, if a mutation that benefits the cell and it is duplication…it will stay that way and not revert. Example… ames reversion assay |
|
|
true reversion
|
true reversion mutation is when it goes back to the wild type
|
|
|
Pseudo reversion or second-sit reversion
|
where these is a mutation somewhere along the dna that makes the phenotype normal again but is not like the original/wild type. It’s a second mutation that creates the same function as the original.
|
|
|
Conditional mutations
|
lost of function in one condition… classic example is the temperature sensitive growth.
|
|
|
point mutation
|
A point mutation that gives the most phenotype mutation is the 2/3 frequency because the triplet code has a wobble code that is readable by two Trna and the first two is always the most important. Those that does not give a phenotype mutation is silent.
|
|
|
duplications
|
now we have duplications where it goes from ab cde then jumps down to cde so there’s two cde’s…duplications of. Types of duplication mutation might not have mutated phenotype…we just increased the copy. can occur as high has 1/10^3
|
|
|
mutation
Lagging strain |
for some reason, the single strain binding protein doesn’t attach the single strain so the dna becomes naked and it could form a loop and the replication polymerase might skip the loop; therefore,…there’s a deletions of dna… a slippage event
|
|
|
Mutagens
|
chemicals that increase mutation rate by modifying the nucleotides in dna. The modification may cause or alter base pairing. However, damage can be very server that it creates an error-prone DNA synthesis (originally called error-prone repair)
|
|
|
Such chemical that changes base pair:
|
Hydroxylamine
Alkylating agents/methylating agents UV O2...radicals |
|
|
Hydroxylamine
|
---change bp from A to G.
induces mismatch repair |
|
|
Alkylating agents/methylating agents
|
add Methyl groups to amino acids (G or T )… ex. CH3 gps to G or T… some block dna systhesis… which is worst than bp changes.
Induces adaptive repair/response |
|
|
UV
|
also block dna synthesis Will induces Pyrimidine dimmers… depending on the organism… (T-T, C-T, or C-C ).. crosslink … it creates a bump where polymerase 3 can’t get over.
Induce nucleotide excision repair |
|
|
O2
Rxn oxygen species (ROS) |
too much is a bad thing because it react with metal (iron) to from hydroradicals
02 radicals are superoxide ….. there are bacteria that change superoxide to oxygen. O2 and OH radicals are ways for the cells to combat against invading bacteria. The macrophages. They are toxics because of the radicals…especially OH radicals being that there isn’t much things to neutralized it????????. There are enzymes that mutate superoxide…they turn the superoxide back into oxygen or react it with water to make more water. They are dangerous to DNA and bad for proteins and lipid. They are really bad for dna because it binds to it and break the backbones (negatively charged)…creating a nick… it breaks the phosphor diester bonds….occur from OH radicals A nick will site there until a replication repair comes my and fix it… in the mean time…the chromosome is broken. Oxidation also created oxidated bases… 8-oxo-G…a common lesion. it will create a mispair. Bp C with T. There are a lot of repair for 8-oxo-G. Such repair includes Nuclexc. Rep (NER) and base excision repair (BER). |
|
|
Screen
|
A screen is when you mutate a culture… but you don’t have a phenotype…death is not one. You have to screen through a bunch of cells. Ex. EC growing in Gal+, you would mutate the Gal and look for Gal-. would give them a plate where there’s a masterplate and plate them on two plate with gal- or gal+.
Another way is used fermented carbon sources…color screening. Plates with Peptides as Carbon sources [limiting] + high [sugar (galactose)] pH indicator. Example…all the colony that can ferment MacConkey (screening media)…all that can ferment sugar turns pink. Fermentation creates acid byproduct…it changes the color. Screen… we have to look at lots and lots of cells. 2. screen – all cells grow but can distinguish some phenotype. Testes we can use are color…. Mac-lac…. Can look for the mutation that’s why. Can only screen 200 colonies/plate. |
|
|
selection
|
only mutants grow... of the 10^10, only one colony could grow.
A selections is nicer…a situation where only the mutation can grow. Ex. a forward mutation screen. A lacI has lacZ, y, A. Lac I…lactose operon… a repressor… it binds lactose which releases the lacZ; however, in the absent of lactose, lac I represses Lac Z in the absent of lactose. If lactose is around… it’ll be unable to bind to the dna (lacZ). Lactose have to bind to lac I in order for lac I to release LacZ. Lactose is an inducer that modify the regulator which increase gene expression LacZ…(b-galactosidase) it takes a sugar (lactose) and breaks it down to glucose+galactose. 12carbon to a 2 six carbon sources. Enzyme is coded by lac Z genes. Jeffery Miller, realized that he can do a selection for mutation in the lacI genes. Instead of giving cells lactose, he gave them a related sugar called phenyl-b-galactoside (PG)…where phenyl-b-galactosidase it can be transported like lactose and can be broken down by B-galactosidase and serve as a carbon source, but it can not induce/bind to Lac I. So you take cells and feed them phenyl-b-galactoside. The wt can’t grow because the cell can’t turn on LacZ. Because they can’t bind to LacI. What he got was a mutation of Lac I. depending what mutation he used… different mutation gave him different results. Why is this nice…this is a selections because it selects only Lac I. Growth on PG…. Wt 1/10^9 Wt+mut 1/10^6 to 1/10^7 If you mutate any repair system then you increase the mutation of the cells A selection is very a powerful way…. Sometimes, you don’t have a selection and will have to screen through many plates. We can narrow it down by using mutation… but when we find the mutation, we have to separate them out and find the right mutation that we want. Why selection? Ex. Let’s say we have a genome and their genome size is 5x10^6bp. On average, on gene is 1,000 bp. Let’s say there is a random change that we’ll get a mutation on any chromosome. The probably of getting a mutation in the target gene is ….1 in 5000. The problem with 1 in 5000 is that about 90% of base substitutions are silence. They can be silence for some reason. Some doesn’t change the amino acid and some doesn’t effect amino acid that are critical and some are non coding region. That means that 10% have a phenotype…. That comes out to be 1/ 5x10^4. Let’s say we can test 200 colonies per plate (screen)…we would have to test 200 plates to find one colony. Testing 200 plates is time consuming and expensive. Now if there’s a selection where we can plat 10^9…we would have multiple colonies. One of the things we can do is increase the mutation in the colonies. It would be easier if we can mut. the cells before we plate and it will increase the mutation by 100 folds. It’s hard to go pass 100 folds because we’ll have a lot of death. With this, we’ll only need to go over 2 plates. Mut can help you, but it can also be hard in the end because you don’t know if it’s the right gene that you want is mutated. |
|
|
The miss match repair system consist
|
Miss match repairs A vs. G, they are big nucleotides and creates a bump and the system will find the bump; however, C-C mismatch…it cant see it.
Some system are very smart in that it can recognized new and old dna strains. In e.coli, they have dam… used of methylation to tell what’s new and old strains. The miss match repair system consist of: MutS – detects the mismatch Mut H – will run across and look for the closes GATC… it’s the one that recognize the bad strain. Mut L – helps the whole process of Mut H and MuT S Pol I – protein that fills in the gap/ also called repair polymerase Dam – Dna ademine methylase…it’s job is to methylate ademine and there are GATC site. There are times when there’s only one strain that’s methylated…called hemimethylation. The strain that’s most likely to suffer mutation is the new strain. let’s say that the replication came through it created the miss match and the error proofing blew pass and didn’t see it, MutS will then detect it MutH then run along the dna and look for the closes GATC site, it with then interact with MutS. They will interact with each other…forming a circle. MutH will transfer the information to MutS on what strain is methylate and Muts will cleave the strain that’s not methylated…it is remove it by a helicase. We’ll now have a gap and DNA pol I will scan the dna and see the cap.. the mistake of pol 1 is 1/ 1mil |
|
|
Nucleoexclusion repair (NER)
|
where one of the pair has a T-T sitting next to each other. Say it belongs to an animal in a UV bed too long and suffer from a T-T permadine primers or C-C…w/e. NER/BER-
System required Uvr A Uvr B Uvr C – endonuclease Uvr D – helicase UvrA and UvrB, together, scans the gene for a specific bump/shape in the dna. They attract Uvr C which cuts the strains. UvrD comes in and cut, then Pol I replaces it. It doesn’t matter on a new or old strains because it’s always toxic…when it sees a bump it will repair it. What they care about is seeing the bulge/bump. The UvrD can cute out a few hundreds of bp |
|
|
Basic recision repair (BRR)
|
it can repair oxidized bases and a number of lesions
When looking at the base of the dna, the base of one strains to another are linked. And the damages are to disturb the base on the 7 base. 8-oxo-G can destabilized the bond on the base and now there’s a whole in the DNA. |
|
|
base excision repair (BER)
|
N-glycosylases (take cares of the sugar)…which breaks the ring then calls in endonucleases to cleave the phosphate backbone of the sugar that creates more than a nick. Dna pol I comes and fill in the new repair base.
Therefore, The oxygen damage kills the base. glycosylases takes care of the sugar (ribosome). Endonucleases break the backbone. Exdonucleases make a bigger gap and dna pol I closes the gap…. That’s BER. |
|
|
Another repair system
ADA – methylation and 8-oxo-G damage |
Where does metylation damage comes from?? S-adenosyl methionine… it’s an energy production. All of our cells have it (indigenous). It’s a high energy compound. Normally it reacts with the appropriate intermediate metabolism compounds but once in a while, it will mistakenly donate a methyl group on the G that shouldn’t be methylated and creates a bulge. There’s methyl transferases that scan the dna looking for the mistake and flip the bp out of the helix. When the pb is flipped out, methyl transferase will methylate itself and take the methyl off the base and onto the protein which kills the protein for it’s activity; therefore, it is not a true enzyme. It’s required over and over again…however, it a major mechanism on taking out methyl groups. G then flips backs in. they do this really really fast.
|
|
|
what happens when there’s a high mutation levels and repair systems can’t get rigid of all the lesions.
|
It can be any type of mutation… these lesions can block dna replication. If replication is block and the repair have been induced for a while but the damage hasn’t been fixed…. The cell eventually doesn’t care and just want to get pass the lesion. So there’s the clamp loader complex that interact with the clamps. The clamps will switch out pol III and put in:
Din B/pol IV Pol V – error prone…cause it doesn’t care…and the result will have a lot of mutation. These can get through all the lesion and the clamp get pol III back on and back to work. However, if the mutation is important then the cells died. translesion systhesis polymerases....where it will replicate over a rang of lesions. We have about 12 translesion systhesis polymerase. Sometimes, it’s more important to get pass a dna then to not have it…humans have a lot of junk dna. |
|
|
ways to isolate mutations
|
selection
screen enrichment |
|
|
enrichment…two categories
|
a. physical
b. Penicillin or d-cycoserine enrichment |
|
|
enrichment:
Physical |
change in the cells. Ex. take bacteria that passes through a filter and take those that didn’t pass though the filter. Alternatively, we have enrich bacteria that are filamentous. We want to do it a lot of times to get the right ones and not by chance. Another example is sticky bacteria…. Grew them on a test tube, pull of the supernatant and take the bacteria that are stuck on the glass. Then test for stickiness on bacteria.
i. In the end, because you’ve gone through a lot of filtering, you don’t know if the bacteria are siblings or if they are unique individual. Therefore, you might have to do a lot of test with different bacteria. think of it as doing physical work. like filtering or growing at lof bacteria |
|
|
enrichment:
Penicillin or d-cycoserine |
Both of the agents disturb cell wall synthesis…the cells will burst and die… but if you take a non growing cell…that’s not making cell wall….then the drug will not affect them. It provides enrichment for the bacteria.
example...... Rules: 1. With each round, we are increasing 1 in 100 each round so we have to do it many times to get the frequency that’s high enough to screen. 2. In the end, starting with one test tube, the end bacteria might be siblings so we’ll have to do it many times. Is there a way to find bacteria under conditions where you’ll have to give them the stuff they need. Start with a culture( 10^8). They allow it to grow on mm+his (or anything we want). Any his auxotroph and wild type can grow. Everyone is happy. They take the tube and wash out the his. They put it in mm w/o His + pen. Any wild type will be growing and synthesizing their cell walls will die. In addition, those that are his- will not be growing and therefore, are not affected by Pen. However, there are some wild type old balls that are flowing around and not growing. Because they are not making new cell walls…they, too, are not affect by Pen and will not die…. About 1%....the other 99% his+ dies Now take the population of cells and wash them out with pen and put them back into mm+his. Everything can grow and be happy. Then you wash it out with Pen… and the other 99% his+ will die while His- will grow. Since we have killed most of His+, we have now enriched his- . as we start off…we have 1x10^8, then 1x10^6, then 1x10^4. Now we can plate the bacteria in mm+ his…everything will grow (about 200). Now replica plate mm+ his-… we’ll probably see 2-4 growth. |
|
|
Auxoptrophs
|
bacteria that cant make cell component (vitamin, AA, NT, etc)
|
|
|
Prototrophs
|
where bacteria can synthize anything they need to function/grow
|
|
|
Multiply mutation and how it can affect the end result
|
An example is a mutation that’s in the same pathway…ex. his operon. There are multiply pathway that feeds into the same end product.
Important to keep in mind that they can affect the same phenotype. -Same pathway (e.g his) -There are many pathways that feed into the same product -Can have mutations that affect physiology…. Slow growth rate…and has nothing to do with his. Another that affect the physiology is the capsule (becomes resistance to many things) Ex. Epistasis…where one mutation phenotype obscures a second mutation phenotype. An example is in the his operon….. HisOGDCBHAFIE. Last step in the metabolic step -----------------------------hol-------------------- His GCBHAFIE D It produces hol and then his. What happen if there’s two mutation…say in G and in D… we’ll have his-. One of them will obscure the other one. Ex. baldness and hair color. If you’re bald, you can’t see the hair color…therefore, baldness is an epistasis of hair color. If there are many ways to mutate a gene…or effect…have to come up with a good way to test that it is the mutation you want. A classic way is complementation analysis. Does your mutation confer the phenotype? |
|
|
epistasis
|
baldness and hair color. If you’re bald, you can’t see the hair color…therefore, baldness is an epistasis of hair color.
experiment..... Late mutation will feed early mutation. This is to figure the order of the enzyme. |
|
|
complementation analysis
Does your mutation confer the phenotype? A classic test is called a complementation analysis |
A classic test is called a complementation analysis… the idea is simple
We have a pathway a b c W-------------X----------------Y---------Z If there’s any mutation at a,b, or c you get Z- Rules: 1. recessive 2. equal number of copies 3. mutation should be in different genes (trans complementation) So you do a merodiploid (partial diploid) where one cell you have two kinds of mutations. If the mutation affects the same gene then there’s no way you can produce product; therefore, you have Z-. If the mutation is in different genes… you’ll get Z+. For this stuff to work, the mutation has to be recessive. It means that the product can function even in the present of the bad gene product. If it’s dominant it means the bad gene product kills the function of the good gene product. Test for dominant is to make one diploid where you have a wild type in one copy and the other copy is mut. We’ve to test one mut at a time. If the wild type is the phenotype we see, then we say the mut is recessive to the wild type. In this case, the cell is Z+. a- b+ c+ //----------------------------------------------------// Recessive End product is Z+ recessive to wild type //----------------------------------------------------// a+ b+ c+ If the cell is Z-, we say the mutation is dominant for the wild type. Why would a bad copy of a gene be dominant of a good copy? Why would a bad gene ruin a good gene? Why would it miss it up? If protein a forms a dimmer, one bad a might prevent function of the rest of the good copy. The mutation must be recessive to the wild type. It is important to have equal copy of the two strains. If one is more…it might overwhelm something and screws up the balance. |
|
|
You are able to mutate a gene through screening, selection, and/or enrichment.
how do you know your gene of interest is what you've mutated? |
To find a mutation…there are many ways
selection, screen, and enrichments (physical and physiological). To narrow down the search, we can mutate the genes we are interested it. However, many things can affect the outcome of what we thing is the gene that we’ve mutated. An example is epistasis. To test for this, we have to do a complementation analysis. To do a complementation analysis of our gene of interest, we have to take three things into consideration. 1. our gene is recessive 2. we have a 1:1 ratio of recessive and dominant genes 3. mutations must be in different genes (trans) |
|
|
Complementation
|
is where we have a gene that can encode the same protein.
|
|
|
cis complementation
|
Lac- …it is a point mutation but acts like a deletions. How… it effects downstream. Or it effected the promoter; therefore, we have to have both the y and z to complement the lac gene… it’s cis. It restores or replace what the promoter can’t do. It restores the promoter region. The mutation has too be co-linear with the gene that needs to be express. Revert the mutation or there’s a second site mutation can create a new promoter that can take over the function of the mutated function.
complement gene has to fill the function of the other gene. Where as the tran is just filling in the missing link. Cis… effecting a site and not the protein |
|
|
tran complementation
|
is where the product on a different gene is insoluble inside a cell that can do the function of the other mutated gene’s end product. The wild type doesn’t have to be co-linear
Tran…means that the mutation is affecting the protein which is flowing around and is insoluble |
|
|
Conditional mutation
|
isolate essential genes where the phenotype can be express in different environments
temperate where one gene can express in on environment but not in another. |
|
|
Informational suppression
|
in the case where there's a stop codon... informational suppression can fix this problem through tRNA error.
Lac Y am…. Am is amber… refer to UGA (stop codon) Ochre….UAA (stop codon) Opal …..UAG (stop codon) The above Lac Y am, ochre, opal……Can be fixed by informational suppression ---------------Lac -------Z-----------Y Review translation. We have a mRNA… we have Shine-delgarno (S-D). Important for translation…it is AG rich sequence. It’s a complementary to a ribosome…s30 Shine – delgarno sequence ( A-G rich) ---------------------SD---------/------Z-----------/--------Y--------/------------- to understand...we have to reveiw translation: Prokaryotic ribosomes….Make up of s 30 and s 50+… together they make up s70… When they come together, they become more compact… therefore, they migrate faster. That’s why it’s s 70 in mass. Kilo darnt? The way translations work is that the small sub unit scan the m-rna for a rna sequence complementation to the rna in the small sub units ribosome. Rna establish small s to start translating. It tells the small unit where to start when it finds the complemented rna and then the big s comes in translate. An equivalent to S-D in euk is a kozak. Ribosome in the big units has important sites on them. After they bind, t-rna comes in. in the ribosome, there's three site, A P and E. the first tRNA come in with an amino acid that has met-f on it. instead of starting at the A, it jumps over to the P. this is what's special about the met f. When there’s nothing on the tRNA (a stop codon), then the P gets unstable and fall out. Back to informational suppression The mutation converts the anti-codon loop to read a stop codon… therefore, instead of reading only a stop codon and adding nothing, it’s can read other codon and adds an amino acid and the ribosome sees that there’s more amino acid coming in so it keeps on going. Therefore… the mutation helps revert the original actual mutation. Would need an amber suppressor to suppress an amber mutation…etc You can check to see if a protein (RNA) is transcribe... if mutation of tRNA does not affect the protein then we know that the RNA is not translated into protein. Not all genes are translated into a protein…for example the RNA that binds to the ribosome…there’s lots of RNA ribosomes…micro RNAs…. They have function but never translated. There’s nothing trna suppressor can do to a protein that doesn’t need to be transcribe. One way to check is to ask if the mutation can be suppress by mutate tRNA genes (only for stop codon) therefore, if the gene does not translate then it informational suppressor doesn’t work. Informational suppressor gives us a way to conditionally suppress a mutation that is defective. If we have a stop codon in an essential function…it will kill the cell. By if the mutation was in a cell that carries a tRNA suppressor (ex. amber) mutation…that mutation will fix the stop codon and the cell will live. Complementation…. One gene might carry the informational suppressor that might help the other gene. It’s a conditional mutation… that help the other gene in the absent of an essential gene. |
|
|
Am is amber
|
refer to UGA (stop codon)
|
|
|
Ochre
|
UAA (stop codon)
|
|
|
Opal
|
UAG (stop codon)
|
|
|
Mapping of mutations
|
-rough mapping – Hfr mapping
-fine mapping – done by co-transduction |
|
|
rough mapping – Hfr mapping
|
Hfr mapping….. use property called conjugation (bacteria matting). Has operon genes that are about 5,000 bp. Rough mapping is it’s at one place rather than another place.
If a gene are not next together…they are probably not an operon |
|
|
Plasmid
|
dna that replicates independently…small chromosome… circler or linear. They are dispensable, not essentials, therefore; ppl have study plasmid and gets an idea of how chromosome are functions and still have the cell function. They replicated independently, they have to have OriP (origin or replication) and They have to have some gene that encode a protein that can distinguish oriP from OriC.
Plasmid tend to encode Rep (dnaA equivalent) to recognized oriP. Plasmid help us mutate genes….cloning…etc Plasmids occurred by conjugation….uses cell to cell contact… very frequent another way is… Transformation – uses pure dna Plasmids are double strained dna |
|
|
OriP
|
origin of replication
|
|
|
R plasmids
|
encodes resistance genes
|
|
|
F plasmids
|
fertility plasmid
|
|
|
Hfr and conjugation:
Tra |
Tra are transfer proteins….they nick the dna strain and pile it off leaving a 3’ hydroxyl end. Then it goes and attach to another set of proteins that creates a bridge and a pore is created
look at diagram in lecture 7 |
|
|
During conjugation
|
During conjugation a single strain dna is given to the new cell. In replication… the chromosomes doesn’t know when to stop so it transfers more dna than what’s encoded. There will be a repeat which carries out recombination which will produce a circle and is now double strained.
|
|
|
rough mapping
|
Hfr.... conjugate with F bacteria and plasmids are paased
plasmids dna unfold or is nicked a tra protein. the tra carries the signal strain dna to the the bridge of what will be call the spores. as the other bacteria replicate the strain, they do in a laggind fashion. The recipient strain will become double. The machine can’t tell when it’s done with a plasmid so it keeps on feeding to the other cell….and we’ll have a repeat. This repeat is able to be recognized. When does it stop? When does the bridge get disrupted? When it gets disrupted… it’s when translation also stops or disrupted. The donor replication cell…is not giving everything up…it keeps one and give another. Conjugation can be a broad range in dna transfer. Part of it is that the pore can be established in a number of ways by different bacteria. Usually a Gm- go into Gm-, but there’s cases where there’s Gm- to Gm+ Also, there’s prok to yeast (euk)…. This transfer is rare but not impossible |
|
|
during conjugation:
Restriction enzymes |
Restriction enzymes recognized the sequence and it digest the dna. They are endonuclease.
One of the things that guide the plasmid survive is the defense mechanism. They have a host restriction or restriction modification. |
|
|
conjugation and restriction enzymes.... we have Modification
|
Modification refers to the methylase that mark the dna of a particular dna as self dna. They add methyl to dna that the restriction enzyme will recognized. Methylases is protecting the dna.
Restriction modification are not as effective. However, a slip though (where a restriction modification didn’t catch the incoming cells) will become a part of the cell and we have evolution. The cell can then modify it with methylase. Why do cells worry about plasmid? There’s much really… but they are protecting from bacteria phage…from virus that might kill them. methylase are thought as immune to virus. Different cells have different restriction dna. The determination of the mating depends on the restriction modifications. They must have the same restriction enzymes. Can think of it as immunity for the cells. |
|
|
selfish dna or selfish elements
|
if we put ourself in the mind of the bacteria dna…we want to make a bunch of us and that’s good because there’ll be more of us. The more of us, the greater success. Over time the cell picked up selective advantages elements. These includes…antibiotic resis. Genes, detox. System for toxic metal (ex. ..hg), detox. System for toluene/toxic aromatic. These plasmids gather the genes that would benefit them.
Plasmids are shelfish and they are interested in their survival. Most encode plasmid addiction modules. They come in different forms. They are genes that encode products that insure that any cells that carry it, it will be unable to get rig of it. The cells carrying the plasmid become addicted to it. How does it work… System CcdA-CcdB…. ccdB is a toxin that inhibit the action of DNA gyrase (relax or supercoil… it makes a nick and reseal the dna). CcdB prevents dna lygation by gyrase. So in the present of the CcdB…gyrase can’t be seal. What it does is encode an anti-toxin, CcdA, that binds to CcdB…and inhibits it. Anti-toxin is unstable…therefore, the cells need the anti-toxin. And CcdB toxin is stable. This prevent the cells from getting rid of CcdB. This is why it’s an addiction where the cells keep the information. The bacteria can fight back by putting CcdA into its chromosome or mutate the CcdB. Also…gyrase can be mutated. The plasmid can spread by being helpful to the cells, or addiction modules, or be neutral. |
|
|
System CcdA-CcdB
|
ccdB is a toxin that inhibit the action of DNA gyrase (relax or supercoil… it makes a nick and reseal the dna). CcdB prevents dna lygation by gyrase. So in the present of the CcdB…gyrase can’t be seal. What it does is encode an anti-toxin, CcdA, that binds to CcdB…and inhibits it. Anti-toxin is unstable…therefore, the cells need the anti-toxin. And CcdB toxin is stable. This prevent the cells from getting rid of CcdB. This is why it’s an addiction where the cells keep the information. The bacteria can fight back by putting CcdA into its chromosome or mutate the CcdB. Also…gyrase can be mutated.
|
|
|
In term of mating...conjugation
|
Donors are F+…because they have the mating mechanism. The recipients are F-. F+ also prevents a F+ from F+ from mating. While F+ x F- are efficient; however, it’s not convenient because they hare big and hard to clone into. It will give you two F+ cells.
in order for the F- to be F+, it has to have everything that the F+ had. Sometime a piece of transposons are able to jump from the chromosome and into the plasmid. An Hfr can put a chromosome into a F- Hfr can’t mate with the same Hfr. However, F- can now gain the chromosome of Hfr Lets say that the donor is a trp+ and the recipient is trp-. Then they can recombine. The transfer is in order and it determines which one transfer first and last. It takes 100mins to transfer one chromosome to another…and this is only done in the lab…and it’s still hard. This means that it’s hard to have this in nature? Ppl do experiments and was used to determine the order in chromosome in bacteria cells. |
|
|
can you transfer chromosomes from bacteria to bacteria?
|
You can transfer the chromosomes but it’s really hard… so you end up with Hfr + F-. Hfr is really hard to convert an F- to Hfr. There are a number of genes to encode the process.
Say if the transfer of a his+ into a his -… we know that the only way to transfer is from the origin because they have his+. However, to become an Hfr, the f have to have all of the chromosome. Therefore it is hard to transfer. |
|
|
mapping using hfr
|
Ask if you start with some hfr strain and cross it with f- recipient and do it for a certain amount of time. What will happen? How much of the donor dna will the recipient get. This is a way where ppl found that dna bacteria is circular. Also…ppl did oriT at different places on the chromosome and find that everything was able to transcribe. We get the same things in the same order.
|
|
|
Rolling circle replication
|
Initiation of replication is independent of host function. In general…there are no dnaA required for plasmid replication. However dnaB and dnaC are required.
Rolling circle replication is where one strain is use as a template and somewhere at OriP there’s a protein that recognize protein at replications and cuts it. Leaving a 3’ end OH. The 3’ end OH is then attached to the double strained origin (DSO). Then Replication goes all the way around and it pushes the outer plasmid. After doing so, we get a final product of one ds and on ss plasmid. Viruses have rolling circles because they are able to replicate plasmid very fast. Fast replication is from the continuous use of the temple circular plasmid. Rep (repercase) puts down a polymerase at the SSO and another strain is replicated. So it makes a dso and a sso. Where sso is polymerase into dso For sso, the ori attract the rep protein, but this time, there’s a priming affect that a primer is place down and we get normal transcription. It required polymerase to make the double and single strained dna. as rep cuts of signal strain one at a time. The template can be used over and over again. It’s efficient to use |
|
|
oriP
|
vegetative ….it replicates all the time. Plasmid have to keep up with the chromosomes….therefore, we have oriP so that there’s duplications when cells divides.
|
|
|
Gene regulations
|
an operon has a signal promoter. The likely hood that the operon being transcribe is determine by the sigma factors.
For a promoter to be active, rna polymerase have to recognized it and melt the sequence. Why?? because rna polymerase will put a complement base to the strain. rna pol melting is done by many factors. |
|
|
Allosteric regulations
|
ex. lacI binding lactose. They are small molecules… sometimes called cofactors that bind to proteins and effects the folding of the protein that changes the function of the protein. Where the protein can no longer bind to the dna. lactose and ipeg are called inducers…because they activate protein transcription
|
|
|
transcription
|
A transcription unit can be one gene with a promoter…and promoter sequence.
It has to have a SD, a start codon and hit one or many stop codons. Every genes has its own transcription signals. There’s also transcription terminator |
|
|
Operons
|
are two or more group of genes that are transcribed from one promoter.
A gene might have one operon but many transcription |
|
|
Signal terminal
|
Signal terminal …has a loop. The structure form within the structure of the rna.
Rna starts at #1 and transcript until it hits a terminal…the rna pol falls off |
|
|
Translation:
start and stop codons |
We can have say an ATG start codon. The ribosome translate and it stops when it hits a stop codon. However, translation picks up again at the AUG using the a of the uaa (stop codon). There’s an overlap of a start and a stop codon. This allow the cells to coordinate the amount of translations.
They do this because of the s-d. it loads the ribosome…it travels down stream and when it hits the start codon… it will translate until it hits a stop codon. when it hits the a in stop codon like AUG…it’s start translating again. And stop when it hits the stop codon. Instead of the ribosome falling off the dna after hittin the stop… the ribosome is able to hover over the stop/start as it goes through the dna. they do this so that they don’t need a lot of s-d. they want to insure that there’s 1:1 relationship. If you have the same ribosome translating the same dna, we will get the same 1:1 ratio whereas if the ribosome are translated separately and required more s-d…chances are we wont get a 1:1 ratio because the ribosome might not be able to attach to the s-d as good as the first. |
|
|
A regulon
|
is where they are turn off and turn on at the same time as the operion…they react with the signal of say…a cAMP. Regulon are regulated at the same time by the same molecule.
|
|
|
A bacterial promoter
|
+1 is the base on a messegnar RNA
-10 base is the pricbnow box About -14 to -17 is a – 35 box sequence |
|
|
prcibnow box
|
About -10 from the base (+1 is the base) of a bacteria promoter
Has similar to the TATATA box. It’s AT rich. Not highly conserved. Also called -10 sequences. It’s -10 because 10 bp from the +1 |
|
|
Rna polymerase
|
large protein encoded by rPOA (2 alpha protein), rPOB (beta sub and prime protein) and rPOC omgea. They are located near the origin.
2 Beta sub and beta prime makes up the body and activation |
|
|
Apoenzyme
|
when you add DNA template and rNTP…it will polymerase blindly…not specific to a sequence.
If add sigma factor… they bind and recognized -10 and -35 on the promoter. Particularly -10 |
|
|
sigma factors
|
sigma 70
sigma 32 Sigma E Sigma 54 (n) Sigma S |
|
|
sigma factors:
sigma 70 (kilodault) |
it’s the house keeping sigma. It the sigma factor that recognizes the gene needed to transcription. Ex. dna polymerase, cytochrome protein important
Sigma change and recognized different sequences…like -35 |
|
|
sigma factors:
sigma 32 (smaller sigma factor)…. |
Recognized the heat shock/stress… the optium temp is 37 C. if shift to 42…the protein will unfold. Therefore, there’s chaperone that help the cell to not unfold… these proteins has sigma 32 on their promoter.
|
|
|
sigma factors:
Sigma E |
Sigma E….recognized the genes that involve envelope stress. Where the bacteria is in stress
|
|
|
sigma factors:
Sigma 54 (n) |
Sigma 54 (n)…involved in Nitrogen regulated genes…. especially in nitrogen fixation bacteria.
|
|
|
sigma factors:
Sigma S |
Sigma S - when genes are turn on when cells are starving… starving response… also turned on in the stationary stage. But more commonly related to the starving of the cell for energy… or carbon.
|
|
|
holoenzyme
|
Rna polymerase with a sigma factor
Different rna polymerase are made with the same structures with different sigma factors. These put together is know as holoenzyme. |
|
|
Canonical promoter
|
-35 box sequence – it’s a 6 nucleotide in a row… the better it is near the concences sequences… the more efficiently rna poly are able to bind and will be able to transcript.
They want to be around 17 bp to have good function… too far and short is bad from -35 to -10 is on all the time… it only needs a rna polymerase and a sigma. |
|
|
bad -35 sequences
|
Many have badly -35 sequences where polymerase bind badly and need activator proteins to stabilize inactions between rna poly and promoter. It helps stabilized the promoter from falling off. The alpha domain sits out in the back and they make contact with sites sitting up stream.
For repressed genes… they have good promoters; therefore, they need a repressor to block transcription. |
|
|
two types of host ranges
|
board and narrow
At the transcription and replication…the virus needs a lot of help from the host….ex. ribosome…pol… cofactors, energy, polymerase... Some of these requirements are specifics… some cells will have everything, some cells don’t. This is where another host range determination can take place. They have to have a specific host factors or else virus might not be able to replicate. |
|
|
host range:
narrow range |
A narrow range is limited to bacteria…and can only be infected to bacteria with little strains differences. It limits the virus to grow on many different bacteria.
|
|
|
host range:
board range |
board range…it can infect bacteria distantly far from each others. The ability to infect different bacteria is important in horizontal transfer.
|
|
|
background of bacteriophages
|
they are the most common entity on earth…. 10^31. there are 10^6 of bacteria per mil and 10 x that amount for virus.
100 folds in virus per mil during algae bloom. 10^31… the different types are less than the number. There’s about 100,000 to 1 mil. Hard to count. half of their genes are devoted to making new structures. They carry polypeptides. they contain tails… and are different from each others. To see how related they are, a proteomic tree was done… where proteins where compared. |
|
|
why is bacteriaphage important to the environment
|
10^31 is a lot and they infect bacteria and lysis them. What we get is a release of bacteria parts….ex. carbon, nitrogen, phosphate… this is a release of nutrients into the environments. It’s is 25% of C cycling in the oceans. There’s a lot of carbon… and it gets recycled by the virus. This effect us directly.
|
|
|
Horizontal gene transfers
|
Transfer of genetic genes from one cell to another. It’s important in health because it affects us.
it affects us in that bacteria are able to transfer drug resistance genes |
|
|
types of viruses
|
We can divide virus into four types. They are similar to euk.
There are DNA…. Double and single RNA….. double and single. dsdna are the most out of the four classes. |
|
|
two types of virus life style
|
These virus have two different life style.
Lytic lysogenic |
|
|
how do we know we have virus and how can we check to see if they are there
|
one way is by looking for it by… taking a sample you think there might be virus and stain them by cyber green? We’ll probably see something big and something small. Bigger things are bacteria and smaller things are viruses. This is a rough way of counting viruses because there might be random things. You’re staining nucleic acid. Another way is by
We can plague viruses… if virus grows inside a cell, they will produce more of themselves. If they can infect neighboring cells…they will. They will defuse and infect. Eventually, the cells on the plate will run out of carbon source and the virus will stop growing as well. They are obligate parasites… if cells doesn’t grow, they cant spread and grow. What we end up seeing is a plague on the plate. By doing titrations, and counting the plaque we are able to count the amount of virus. |
|
|
virus.....eclipse
|
the minimum amount of time that takes infection to disassemble and reassemble new virus particles
|
|
|
virtual infection
|
1. absorption ( this is reversible…on and off… then on …until you get irreversible commitment).
2. irreversible attachment - After the irreversible attachment, the virus inject it’s dna into the bactera 3. injection or other means of DNA entry ( in some cases virus manage to push in enough of nucleic acid into the cells, the nucleic acid has a promoter at the end and rna polyerase recognizes the promoter and starts transcribing the gene… the bacteria actually sucks the virtual nucleic acid into the cells). 4. transcription ( to get to mRNA then to proteins) and replication of nucleic acid. Proteins are assembles into new structures and the neulic acids are package into the structues 5. packaging 6. host cell lysis… at the end where the virus grows, it needs to get out of the cells by lysising it….most of the time. at the end, virus multiply and lysis the host cells to get out. |
|
|
virtual infection:
absorption |
1. absorption ( this is reversible…on and off… then on …until you get irreversible commitment).
|
|
|
virtual infection:
irreversible attachment |
2. irreversible attachment - After the irreversible attachment, the virus inject it’s dna into the bactera
|
|
|
virtual infection:
entry |
3. injection or other means of DNA entry ( in some cases virus manage to push in enough of nucleic acid into the cells, the nucleic acid has a promoter at the end and rna polyerase recognizes the promoter and starts transcribing the gene… the bacteria actually sucks the virtual nucleic acid into the cells).
|
|
|
virtual infection:
transcription |
4. transcription ( to get to mRNA then to proteins) and replication of nucleic acid. Proteins are assembles into new structures and the neulic acids are package into the structues
|
|
|
virtual infection:
packaging and lysis |
5. packaging
6. host cell lysis… at the end where the virus grows, it needs to get out of the cells by lysising it….most of the time. at the end, virus multiply and lysis the host cells to get out. |
|
|
what does a virus look like
|
It has a capsid/head. They can be prolate (long), oval shapes
Tail…is attached to the head by a collar. The tail is inside a contractile sheath where there’s a tube inside the virus and the outside tail can squeeze dna. Tall fibers… they attach bacteria. The tall fibers have specific recognition to bacteria’s surface. They have specific receptors to different proteins. One way to determine host range is by the tall fibers. Tail fibers also have proteins that can digest piptidglycan. It is the most rigid structure and has has to get its dna through the bacteria. Virus doesn’t release or goes into the cell of the bacteria. However, nucleic acids are needed to be injected into the bacteria. |
|
|
Lytic cycle of phages
|
There are about 40-50% that carry out a life cycle that is only lytic
Start with a bacteria cell with its own chromosome. The dna is injected inside the bacteria. Few proteins are injected. Frequently… dna are circularized. Phage dna most of the time are linear dna. Dna are package into the newly virus as linear molecules. And they are package. The cells then lysis. And release virus How do viruses know when to lysis. If they lysis too early…. ATP synthesis will be lost. In order to make 100 newly virus…u need a lot of ATP. Disruption of the cells are membrane atp syntesis. How long is a generation of bacteria cells… 20-30 mins. Therefore, the virus needs a lot of ATP. The burst size ( = number of viral particles or virons released per cell) can range btw 30-1000, depending on the virus and on the growth conditions of the host. The poor the carbon source, the weaker the burst size Phages are resistance to the environments. |
|
|
Lysogeny
|
We start with a virus that infects a host cells. The virus can sense if the bacteria is in a good or bad environment. That decision tells the virus to go through a cycle to produce more virus or to go into the host chromosomes. They integrate and the viral genomes where virus will divide along with the bacteria. We get a lysogen because the virus can lysis the bacteria any time in the future. Prophage is a procurer of a phage.
|
|
|
Prophage
|
Prophage is a procurer of a phage
In the prophage state, they have a repressor and a gene that intergrate the virus dna into chromosome known as integrase. They can live on for a long time if nothing happen to the bacteria/host. 50-70% of a bacteria carry a prophage. |
|
|
When do the lysogeny become lysis
|
when there’s dna damage. Ex. if there’s a mutagen or uv damage. They have a switch that decide when they are safe. In the case of a uv, the virus are more resistance by themselves than in a host cell…we then get:
|
|
|
Excision
|
Excision of the phage…they become themselves again. where the get out of the bacteria chromosome
|
|
|
intergrase
|
. Phage have a protein called intergrase and intergrate. They scan the chromosome for a particular site…ex. att B (bacteria site) and att P (phage site) ( att= attach). P and b pair up and recombination takes place. Intergrate will cut at the attt b site and reunited to att p . we get intergration of the phage to the bacteria and get a big circle.
From att p and attb…we get att L and att R |
|
|
excisase???
|
In order for att L and att R to combine…it needs excisase. if the virus decides that they are not happy, it will increase intergrase and make excisase and att L and att R will take itself out of bacteria chromosome. Virus can then replicate and grow.
|
|
|
decision for lysogenic virus
|
They have to shut off viral protein synthesis and not lysis their host
There’s a lysogenic switch… they repressor the protein…called C1. and transcript from . the promoter PRM. The genes are transcript both ways by two promoters. There’s two promoter going left and right required for the phage cycle. Except for the repressor which has its own promoter. When the phage want to stay as a prophage. The repressor binds to the operator left and operator right. An operator sequences where repressors bind. In the case of the phage repressor, they are down stream o There’s dimmer binding of the Ori and we get repression. The dimmers talk to each other by a protein-protein interaction. In that a loop is form and is stable. The repressor does not fall off by accident. You need the protein to be stable and it is only stable when there’s rich minerals and/ or when the environment has low cAMP. In bacteria… the cell is growing when there’s low cAMP. |
|
|
dimmer binding of the Ori and we get repression
|
There’s dimmer binding of the Ori and we get repression. The dimmers talk to each other by a protein-protein interaction. In that a loop is form and is stable. The repressor does not fall off by accident. You need the protein to be stable and it is only stable when there’s rich minerals and/ or when the environment has low cAMP. In bacteria… the cell is growing when there’s low cAMP.
|
|
|
low cAMP
|
low cAMP is similar to High ATP (virus need to grow). Low cAMP = high protease which cleaves cI (repressor); therefore low repressor creates lytic
|
|
|
high cAMP
|
Low ATP or high cAMP… there’ll be low protease and high CI and lots of lysogency
|
|
|
packaging:
|
Generalized transduction
Specialized transduction |
|
|
packaging:
Generalized transduction Generalized transduction |
Generalized transduction….where it fills its cap with as much chromosomes as it could. Those particular are called transductining dna… only their DNA. The only thing that’s present is chromosome dna
|
|
|
packaging:
Specialized transduction |
Specialized transduction – Virus can package bacteria dna as well. This might or might not affect the growth of the virus.
Ex. vc must have to carry a prophage that has the toxin. Cholera toxin genes…. Is carried on a prophage. If VC carry a prophage… then we get Cholera. |
|
|
Lysis vs. Lysogeny decision
|
1. at two different time in the cells
a. at the original establishment of lysogeny b. to remain a prophage or to bail and replicate as a phage |
|
|
Lysogeny decision
|
Phage has been injected into the cells as a linear and has a sticky ends… the ends comes back and form a circle… happens very fast upon infections
Places on the chromosome…att P (attachment)… use to intergrate/ insert the genome into the bacteria. Also, region called the immunity region consist to two promoters ( PR and PL). Together with att P and Ihr, they interact and carry out integration, by doing a dna exchange. Att B cuts and reunites with att P. Attp and attB attach to each other. |
|
|
Recombination function
|
Protein called N, cro, O P Q
Recombination function CI is the master repressor Cro is also a repressor... c1 and cro...battle it out and determine what is lysis or lysogeny cII is an activator of cI gene and also a sensor of the metabolic state of host cells O is DnaA analog to bacteria cells P is DnaC analog to bacteria cells OP is silences and only fire if only the O gene can recognize it and recruit the P genes and the P gene steals the dnaB helicas of the host and brings it to the phage origin of replications. When we’re talking about the phage replicating, in the circular stage… where the gene is now independent)… it’s when the module comes to play. Pass Q… all the genes needed to make the survival of the bacteria…like tails..etc. Once assemble… the virus needs to lysis the cells. |
|
|
Terminase
|
package the dna into the head of the virus.
cII is involve in lysogency. Int is also in lysogency |
|
|
xis
|
helps the lytic phage because it is required to go backward. Intergrase by itself can not do combination btw att L and att R. it requires intergrase, xis, and ihr.
|
|
|
N
|
anti-termination protein…. Where it would discard the tr1 and tl1
It directly modify rna polymerase after it is transcribe so that rna can ignore the terminals (tr) |
|
|
Cro
|
also a repressor... c1 and cro...battle it out and determine what is lysis or lysogeny
If we have a burst of cro at the beginning, the promoter for cI will be shut off. Before that happens, N is also being transcribe. N will allow both terminators to go through where we will get production of CII proteins. is a small protein and forms a dimmer… that binds to Or3 or Ol3 first (it’s there preference)… if there’s a lot of cro around, they’ll fill in the rest. When cro fills in or3, it is also repressing the Prm that transcribe CI. Cro is made early and the real lysogeny is cI. They will fight with each others for dominances at the operator sites. Which ever one wins will determine the fate of the phage. If Cro wins…CI level goes down If CI wins… CI level will go up…. They also activate its only transcription If you look at the two proteins, they both form dimmers. Cro and cI both form dimmer. cI is more complex than cro. cI has a dna binding domain that is analogist to Cro, but they like different site of the protein….like CI likes to bind at the first site 1, while cro likes to bind at 3. |
|
|
If N is made
|
we’ll have CII…what ever happen, CII will decide the fate of the phage.
cII is a positive activator of cI transcription . if cII is stable…it will drive the phage to lysogeny cause we get cI production. How do you regulate cII…. With cAMP. |
|
|
Low cAMP
|
means high ATP… hi level of FtsH/HflHfl (high frequency of lysis) protease. cII unstable and will
|
|
|
Hi cAMP
|
low ATP, low ftsh/hfl protease CII will be stable and will go to lysogeny.
|
|
|
Protease and responds to energy level
|
metabolic state. If cells is in high metabolic state… then protease will be active and there’ll be unstable cII. And cI will not be produce. This would equal to a lot of virus.
If the infected host is healthy and is replicating well…that means that there’s a lot of bacteria and the virus can infect the other host. If there’s not a lot of host, there’s not point in making them. If the host cell is not replicating well…. There wont be a lot of cells to infect and there wont be a lot of protease. In this case, the phage would have a stable cII and be lysogeny. |
|
|
Prophage replicate....
|
The virus are replicating passively
|
|
|
lysis....mode??
|
it is actively replicating
|
|
|
CII
|
is a critical sensor to the two steps
activates PRM and also PI (it needs integrase)… needed In order to intergrate into the host genome. involve in lysogency. Int is also in lysogency |
|
|
CI
|
CI is the master repressor
If CI wins… CI level will go up…. They also activate its only transcription If you look at the two proteins, they both form dimmers. Cro and cI both form dimmer. cI is more complex than cro. cI has a dna binding domain that is analogist to Cro, but they like different site of the protein….like CI likes to bind at the first site 1, while cro likes to bind at 3. When there’s a lot of CI, it binds and form stable dimmers and head for 1. when it fills one, it is highly cooperative with itself and will fill in the second site as well. We’ll get protein-protein interaction. A loop is created when they come together with the dimmers. The loop includes Or and Ol. A lot of CI wins the fight against cro because they are very stable. They are very effective once the sites are filled. If cI is made, it shuts off pL and pR excepts for CI. CI binding at the or1 site and activates it’s own promoter. It is an auto regulator protein. It does this by recruiting rna polymerase and stabilized rna polymerase on the dna. At the same time…shuting off transcription of other proteins |
|
|
to remain a prophage or not
|
cI repressor is part of a regulon…it’s part of the sos regulon… it is regulated by Lex A. it represses SOS. It’s responsive to dna damages. If cells sense DNA damages… there’ll be a high increase in single strains dna. Cells have a lot of signal dna strains is exposure to UV. It will crate permadine dimmers and there’ll be a bump. the bump will have to be fixed by the polymer where it’ll nick the dna. Also, okazai fragments…where there’s signal strained… however, there’s not much around until there’s damages from UV. this damage will increase signal strained dna. These dna are bounded by recA. RecA function is to repair broken dna. When recA binds to ssDNA, it forms recA* (activated). recA* interacts with lexA and cI repressor. If recA* touches the lexA repressor or CI, these repressor will unfold a little bite.
If recA interact with lexA or CI, the hinge will unfold and reviews a cryptic ( where normally, the site is inactive. It is hidden where there’s a hidden protease and only reveal when there’s a change ) site. If they interact with recA*, the protein dimmer gets cleaves and the protein-protein interaction domain is no longer able to hold the dna binding domains together and it makes an ineffective repressor. The repressor falls off the dna and no longer represses. Prophage responses to dna damage at the same time the cells response to dna damages. This is when the virus wants to get out. cI dimmers cleaves themselves if there’s dna damage…the repression loop falls apart. Pr and Pl are strong promoters and will transcript whenever they can. Int xis are made and recombine the chromosome out of the bacteria. Dna damages is an induces sensor if there’s dna damage…the repression loop falls apart. Pr and Pl are strong promoters and will transcript whenever they can. Even if the cells is growing poorly… as long as there’s recA*, CI will continuous to cleave any cI repressor. recA* doesn’t cleaves cI repressor, but it activate cI to cleave itself. And the same for lexA repressor. A lot of phage dna are made as well as structures. We also get terminase that package everything together. Why is it good to make everything when the host is damage? Because the dna are protected in the capsule of the virus. It’s better to be free floating then not. Establishing lysogeny… pr and pl are active originally and then they are shut off. If the phage wants to integrate and make N but not xis. It makes a little bit of N so that if their dna pops out, they want to pop back in…..we’re gonna talk more about this on Monday. |
|
|
Lex A
|
These dna are bounded by recA. RecA function is to repair broken dna. When recA binds to ssDNA, it forms recA* (activated). recA* interacts with lexA and cI repressor. If recA* touches the lexA repressor or CI, these repressor will unfold a little bit.
If recA interact with lexA or CI, the hinge will unfold and reviews a cryptic ( where normally, the site is inactive. It is hidden where there’s a hidden protease and only reveal when there’s a change ) site. If they interact with recA*, the protein dimmer gets cleaves and the protein-protein interaction domain is no longer able to hold the dna binding domains together and it makes an ineffective repressor. The repressor falls off the dna and no longer represses. Prophage responses to dna damage at the same time the cells response to dna damages. This is when the virus wants to get out. cI dimmers cleaves themselves recA* doesn’t cleaves cI repressor, but it activate cI to cleave itself. And the same for lexA repressor. When recA* interact with the repressor, they change conformation and they reveal the protease activity. The protease gets activated and starts to cleave. The repressor falls over and transcription happens. Dna damage is an inducing signal |
|
|
PL
|
If PL gets activacted… int and xis are made. These two recombine the phage out of the bacteria chromosome and establish a circular dna. the Pr promoter activate the replication function and activate all the structure genes. Replication gives up more phage dna. also the gene replicate by rolling circle.
|
|
|
There are two places were you can get regulation.. without activator or repressor
|
cells have anti-sigma factors. They bind sigma and keep them from binding to polymerase… ex. flgM…. It interact with sigma 28 (important for transcription promoters up stream in the motility genes) the anti…sequester…sigma 28 which is important for motility genes ( this genes is all over the DNA and is 1% of coding capacity… also make flagella. Flagella has long protein made of flagellin… in order for flagella to function correctly, the cells need a function of structures.
Therefore, there’s flgM. And the cells get rid of flgM after the flagella is fixed. |
|
|
Rpos (sigma s)
|
starvation responses… anti rpos (rsd)
|
|
|
abortive initiation
|
Weak promoter can be weak at the +1 region or -35. what happen is that as polymerase start transcription, the polymerase can fall of, bind then fall off….this is called… abortive initiation. They make small mRNA of about 6-8 nt.
The weaker the binding of promoter… high abortive initiation If it can escape the abortive initiation, we get longer chains. |
|
|
Polarity
|
---------------------SD---------------/-------B-------/-------A----/----D---------
Let’s say in the A gene from diagram above… it hits a stop codon, the ribosome falls off. If there’s a new stop before the initial stop…then there’s going to be naked mRNA. The space will not be cover by mRNA On the naked mRNA, there’s Rho (rna helicase). It will travel and find the dna/rna complination. Rho takes the two apart through disruption RNA-DNA duplexes. This stops transcription. It stops transcription when mRNA is naked. This is an unnatural situation because transcription and translation occur at the same time, the are coupled. stop mut… will stop the translations. If translation stops within an operon…almost always the transcription down stream will also be blocked. Polarity… that a mutation at A is polar to the downstream. Polar mutation is that it block the transcription of the gene downstream. In the case of a mutation |
|
|
polarity:
Rho |
On the naked mRNA, there’s Rho (rna helicase). It will travel and find the dna/rna complination. Rho takes the two apart through disruption RNA-DNA duplexes. This stops transcription. It stops transcription when mRNA is naked. This is an unnatural situation because transcription and translation occur at the same time, the are coupled. stop mut… will stop the translations.
|
|
|
cells and virus...dividing
|
As cells divide, the virus divide… therefore, the concentration drops… up and down.
If you have a concentration of repressor go down... you want a high cooperative |
|
|
intergration
|
from virus plasmid to chromosome....
it needs int (egrase) + IHF (helper) |
|
|
excision
|
+int (egrase)
+Xis +ihf |
|
|
sib
|
In the prophage….starting at Pl and since there’s no sib…int and xis is able to produce a lot.
Why doesn’t Pi terminate before int. N is the anti terminator. It modifies rna polymerase at Pl and Pr. Part of the attL site is the terminator. Pl can go through the terminator while Pi cannot. Therefore, it can only make int because of the gap…. Sib…why is it important….if the cell doesn’t want to integrate; they don’t want to make int and xis Attracts RNAse III which cuts which cuts sib |
