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Flashcards in Stress Response Deck (50):
1

sigma S

- 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

Specific stressers modulated by sigma S

- temperature (heat, cold)
- oxidative stress

3

sigma factor's role in transcription

- recognize the promotor and help RNA polymerase bind to the DNA.

4

regulation by sigma S

- 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

regulation of sigma S

- regulated at the transcriptional and translational level.
- only transcribed until the bacterium senses stress.
- three promotors for rpoS
- each promotor controlled separately

6

Transcriptional regulators of RpoS

- 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

Three promotors of RpoS

- one directly preceding the gene
- one found within the upstream gene
- one upstream of the preceding gene.

8

Translational regulators of RpoS

- 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

Stem loop and spacer of DsrA and OxyS

- will melt out certain regions
- DsrA turned on by heat stress
- Oxy S under peroxide stress will stabilize

10

E. Coli sigma S regulation

- 481 directly regulated by sigma S
- 100 more moderated by sigma S
- 10% of genome

11

Starvation

- 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

ppGpp

- 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

cAMP

- 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

adenylate cyclase

- catalyzes the conversation of ATP to cAMP and pyrophosphate

15

carbon starvation regulon

- alternative carbon and energy utilization pathways
- encode carbohydrate transport and catabolic operon like those for lactose, arabinose, and maltose.

16

targets of ppGpp

- 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

affected due to change in specificity

- rRNA genes are down regulated
- Amino acid biosynthesis genes are up regulated
- CAP synthesis is up regulated
- RpoS is up regulated

18

Heat shock and protein folding

- 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

if protein is too cold

it will lose flexibility

20

if protein is too hot

- it will denature
- if denatured too badly, it will aggregate with other proteins through hydrophobic interactions.

21

Heat shock response

- 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

RpoH

- 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

Targets of RpoH

turns on three classes of genes:
- holdase chaperones
- foldase chaperones
- proteases

24

Holdase chaperones

- 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

foldase chaperones

- 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

GroEL

- provide hydrophobic environment for proteins to fold, provide energy to find correct structure
- foldase

27

proteases

- degrade unfolded proteins, allowing amino acids to be recycled into new proteins.

28

oxidative stress

- 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

Superoxide

- 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

superoxide dismutase

- detoxifies superoxide
- one molecule of superoxide reduced to H2O2, one is oxidized to O2 - dismutation

31

internal superoxide

- 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

external sources of superoxide

- 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

macrophages and superoxide

- have an enzyme called NADPH oxidase that will specifically reduce O2 to O2- when the cell detects and infection.

34

hydrogen peroxide

- 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

catalase

- 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

Hydroxyl radical

- 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

Why cells die from radical oxygen species?

1. superoxide is produced by ETC
2. reacts with Fe-S clusters of ETC to release free Fe2+
3. Fe2+ attracted to negatively charged phosphate backbone of DNA
4. superoxide disputes from O2- to form H2O2
5. H2O2 reacts with Fe2+ to form highly reactive hydroxyl radical
6. hydroxyl radical reacts with first macromolecule it encounters which is often DNA.
7. Leads to double stranded breaks in DNA, which leads to death.

38

Regulation of oxidative stress response

- main players are SoxRS and OxyR

39

SoxRS

- responds to O2-

40

OxyR

- responds to H2O2

41

SoxR

- inactive in reduced state
- oxidized by superoxide, activates transcription of soxS

42

SoxS

- turns on transcription of superoxide dismutase and endonuclease IV, a DNA repair enzyme

43

OxyR

- 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

alkyl-hydroperoxide reductase

- can fix oxidatively damaged lipids, as well as sRNA oxyS, that interacts with general stress response mediated by sigma S

45

Human disease linked to oxidative stress

1. cancer
2. aging
3. Ischemic reperfusion injury

46

cancer

- oxidative damage to DNA causes mutations

47

aging

eat antioxidants, stay young

48

ischemic reperfusion injury

1. organ is cut off from blood supply. No O2, no blood
2. Mitochondria stop working
3. Mitochondria start to lyse
4. Lysed mitochondria release ETC components (lots of Fe/S clusters)
5. Blood supply is returned (abundant O2, food)
6. O2+Fe=fenton chemistry

49

nitrogen stress

- nitrogen stress can be considered a subset of oxidative stress, as toxic forms of N also contain O

50

peroxynitrite damage

- 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