Stress Response Flashcards
1
Q
sigma S
A
- global regulator of the general stress response
- also called rpoS
- S stands for stationary because sigma S is found in high levels in stationary phase cells of E. Coli
- triggered by starvation that comes after stationary phase
2
Q
Specific stressers modulated by sigma S
A
- temperature (heat, cold)
- oxidative stress
3
Q
sigma factor’s role in transcription
A
- recognize the promotor and help RNA polymerase bind to the DNA.
4
Q
regulation by sigma S
A
- genes recognized by the sigma S version of RNA polymerase have slightly different promotor regions that are only recognized by sigma S and thus will only be transcribed when sigma S is present.
5
Q
regulation of sigma S
A
- regulated at the transcriptional and translational level.
- only transcribed until the bacterium senses stress.
- three promotors for rpoS
- each promotor controlled separately
6
Q
Transcriptional regulators of RpoS
A
- cAMP levels and CAP - glucose starvation
- pppGpp - stringent response of starvation
- NADH/NAD+ ratio - starvation
- Quorum sensing
- Acetate and weak acids - fermentation produces, acid stress
7
Q
Three promotors of RpoS
A
- one directly preceding the gene
- one found within the upstream gene
- one upstream of the preceding gene.
8
Q
Translational regulators of RpoS
A
- default for sigma S translation is off
- very little made because ribosome binding site buried in secondary structure
- small regulatory RNAs bind through RNA-RNA interactions that allow for translation
- Temperature - Heat and Cold - dura
- Oxidative Stress - OxyS - down regulates
9
Q
Stem loop and spacer of DsrA and OxyS
A
- will melt out certain regions
- DsrA turned on by heat stress
- Oxy S under peroxide stress will stabilize
10
Q
E. Coli sigma S regulation
A
- 481 directly regulated by sigma S
- 100 more moderated by sigma S
- 10% of genome
11
Q
Starvation
A
- accumulate two molecules
- cAMP - in response to glucose depletions
- (p)ppGpp - in response to amino acid depletions
- act independently but synergistically to help cell overcome starvation
- cell uses ribosome to measure the pool of charged tRNAs
12
Q
ppGpp
A
- when a cell runs out of charged, amino acids, the ribosome will contain uncharged tRNAs
- when a ribosome unloads an uncharged tRNA into A site, a ribosomal protein RelA, will catalyze the transfer of a diphosphate from ATP to the 3’ OH of GTP, forming pppGpp.
- quickly converted to ppGpp.
13
Q
cAMP
A
- when glucose is present, the level of cAMP is low
- when glucose levels decline, adenylate cyclase is activated
- cAMP binds to CAP and regulates ~100 members of the carbon starvation regulon.
14
Q
adenylate cyclase
A
- catalyzes the conversation of ATP to cAMP and pyrophosphate
15
Q
carbon starvation regulon
A
- alternative carbon and energy utilization pathways
- encode carbohydrate transport and catabolic operon like those for lactose, arabinose, and maltose.
16
Q
targets of ppGpp
A
- binds directly to the beta subunit of RNA polymerase changing specific of promotors it recognizes.
- makes sure cell will stop making ribosomes, will start making amino acids, turn on alternative carbon utilization pathways, and turn on general stress program.
17
Q
affected due to change in specificity
A
- rRNA genes are down regulated
- Amino acid biosynthesis genes are up regulated
- CAP synthesis is up regulated
- RpoS is up regulated
18
Q
Heat shock and protein folding
A
- the structures the various proteins in the cell are determined by their primary sequence, and the correct structure is thermodynamically defined by the lowest energy level achievable.
- within a narrowly defined temperature range. if taken out of that range, it will not behave properly.
- the proper folding of a protein is critical for its function
- make sure they don’t aggregate
19
Q
if protein is too cold
A
it will lose flexibility
20
Q
if protein is too hot
A
- it will denature
- if denatured too badly, it will aggregate with other proteins through hydrophobic interactions.
21
Q
Heat shock response
A
- upon sensing heat shock, the cell will start making proteins that can help it cope with the denatured proteins it will encounter.
- all proteins that will be turned on are controlled by sigma R or RpoH
22
Q
RpoH
A
- gene transcribed all the time, but not translated due to secondary structure that precludes ribosome binding at physiological temperatures
- if temp increases to levels that will require heat shock response (42 for E. Coli) , the temp will melt the secondary structure and allow for translation of the gene.
23
Q
Targets of RpoH
A
turns on three classes of genes:
- holdase chaperones
- foldase chaperones
- proteases
24
Q
Holdase chaperones
A
- DnaJ, DnaA, GrpE in E. Coli
- bind to unfolded proteins and keep them from denaturing and aggregating during the heat shock
- let them go when temperatures fall.
25
Q
foldase chaperones
A
- actively help mis-folded proteins to refold, they use ATP to import energy to misfiled proteins and give the proteins a safe place to refold in their nature structure.
26
Q
GroEL
A
- provide hydrophobic environment for proteins to fold, provide energy to find correct structure
- foldase
27
Q
proteases
A
- degrade unfolded proteins, allowing amino acids to be recycled into new proteins.
28
Q
oxidative stress
A
- oxygen is a valuable commodity because it has a high redox potential, and can be very toxic.
- in order to reduce O2 into the non-toxic water molecule, 4 electrons must be added one at a time to O2, and each product is quite reactive.
29
Q
Superoxide
A
- produced by 1 electron reduction of O2
- reacts with cellular targets and oxidizes them
- favorite targets are Fe-S clusters which release free iron when oxidized that damage the cell.
30
Q
superoxide dismutase
A
- detoxifies superoxide
- one molecule of superoxide reduced to H2O2, one is oxidized to O2 - dismutation
31
Q
internal superoxide
A
- superoxide produced by electrons “leaking” from the respiratory chain.
- If an electron destined for the next electron transport component does not reach it, it can easily be based to O2.
- respiring cells have a much higher concentration of O2-
32
Q
external sources of superoxide
A
- superoxide is produced by immune cells in response to infection
- when infected, they will respond with an oxidative burst of O2- directed at the invader.
33
Q
macrophages and superoxide
A
- have an enzyme called NADPH oxidase that will specifically reduce O2 to O2- when the cell detects and infection.
34
Q
hydrogen peroxide
A
- because it results in the two-electron reduction of O2, it is not a radical.
- Is a powerful oxidant, but when exposed to free iron, will produce the dangerous hydroxyl radical via the Fenton reaction.
- important reaction because Fe2+ is released from Fe-S clusters by superoxide.
35
Q
catalase
A
- detoxifies 2 molecules of H2O2 to water and O2
- 3 different kinds of catalase, depending on the metal (Fe, Mn, or Cu and Zn)
36
Q
Hydroxyl radical
A
- one of the deadliest form of reactive oxygen, will cause breakage of the DNA backbone.
- will also damage protein and lipid
- no enzymatic way to deal tis it. It will react with the first macromolecule it encounters because it is so short lived.
37
Q
Why cells die from radical oxygen species?
A
- superoxide is produced by ETC
- reacts with Fe-S clusters of ETC to release free Fe2+
- Fe2+ attracted to negatively charged phosphate backbone of DNA
- superoxide disputes from O2- to form H2O2
- H2O2 reacts with Fe2+ to form highly reactive hydroxyl radical
- hydroxyl radical reacts with first macromolecule it encounters which is often DNA.
- Leads to double stranded breaks in DNA, which leads to death.
38
Q
Regulation of oxidative stress response
A
- main players are SoxRS and OxyR
39
Q
SoxRS
A
- responds to O2-
40
Q
OxyR
A
- responds to H2O2
41
Q
SoxR
A
- inactive in reduced state
- oxidized by superoxide, activates transcription of soxS
42
Q
SoxS
A
- turns on transcription of superoxide dismutase and endonuclease IV, a DNA repair enzyme
43
Q
OxyR
A
- contains cysteine residues that are normally reduced
- upon exposure to H2O2, cysteine residues are oxidized and OxyR becomes active
- targets are catalase and alkyl-hydroperoxide reductase
44
Q
alkyl-hydroperoxide reductase
A
- can fix oxidatively damaged lipids, as well as sRNA oxyS, that interacts with general stress response mediated by sigma S
45
Q
Human disease linked to oxidative stress
A
- cancer
- aging
- Ischemic reperfusion injury
46
Q
cancer
A
- oxidative damage to DNA causes mutations
47
Q
aging
A
eat antioxidants, stay young
48
Q
ischemic reperfusion injury
A
- organ is cut off from blood supply. No O2, no blood
- Mitochondria stop working
- Mitochondria start to lyse
- Lysed mitochondria release ETC components (lots of Fe/S clusters)
- Blood supply is returned (abundant O2, food)
- O2+Fe=fenton chemistry
49
Q
nitrogen stress
A
- nitrogen stress can be considered a subset of oxidative stress, as toxic forms of N also contain O
50
Q
peroxynitrite damage
A
- will nitrate tyrosine and tryptophan and guanine
- can also oxidize cysteine, methionine, lipids, and may cause DNA modifications and strand breaks
- no enzymatic protection.
- NO + superoxide makes peroxynitrate
