Ls3-Ch17 Gene Regulation In Eukaryotes

Front 1

01: In what step does most eukaryotic regulation take place?

Back 1

TRANSCRIPTION

Front 2

02: If both an activator and repressor are working, which usually wins out?

Back 2

the repressor

Front 3

03: Where does RNA Pol bind?

Back 3

the promoter

Front 4

04: How is the eukaryotic regulatory region different from prokaryotes?

Back 4

it is often very far (thousands of BP) away from the promoter

Front 5

05: How does a regulatory specify which gene it works on?

Back 5

BOUNDARY ELEMENTS (aka INSULATORS): regions that define where a gene is. will have proteins bound to them

Front 6

06: Does transcription in eukaryotes usually require one regulatory element?

Back 6

NO: usually needs lots to initiate transcription

Front 7

07: What helps us learn about gene eukaryotic regulation? One example

Back 7

REPORTER GENE:
a gene whose activity is easily measured.

ie β Galactosidase:
can add a protein which will change color depending on how much of it is available.

if you put in a regulatory region of another gene that will instead act on your reporter, you can see where those genes act.

Front 8

08: What is the structure of eukaryotic regulatory proteins?

Back 8

often DIMERS:

have separate BINDING and ACTIVATION domains

Front 9

09: How can we have variation in eukaryotic regulatory proteins?

Back 9

HOMODIMERS: same dimers.
HETERODIMERS: different dimers together, gives larger possibilities for variation

Front 10

10: Where do eukaryotic regulatory proteins bind?

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ACTIVATING SEQUENCES: can be upstream or downstream. can have multiple binding sites

Front 11

11: what did we learn from the DOMAIN SWAP EXPERIMENT?

Back 11

It showed us that both the activating and binding regions of the regulatory protein are needed.

Activating regions can bind to other binding regions to activate the binding region's gene.

Front 12

12: What are some binding motifs?

Back 12

HOMEODOMAIN: single subunit...esp helix-turn-helix

ZINC FINGERS: charged, will interact w/ negative DNA backbone

LEUCINE ZIPPER: dimer. each part could have different binding domains.

HELIX-LOOP-HELIX

Front 13

13: What is a common mechanism for activating proteins? What is the significance?

Back 13

activator brings in MEDIATOR: holding onto RNA Pol via many NON-SPECIFIC SEQUENCES...means that it can work with several different activators.

mediators are often positively charged

Front 14

14: Is there a holoenzyme for eukaryotic activation?

Back 14

it is debatable, we think it only forms in vitro.

Front 15

15: What is one way the mediator can act?

Back 15

can push the LexA protein (bound to Rna Pol) onto lexA site on DNA

Front 16

16: What is a very important overall eukaryotic activation/repression mechanism? How does it act?

Back 16

NUCLEOSOME REMODELING:

activator protein binds, brings in acetyl transferase, expands chromatin to 10nm fiber

OR:
activator recruits chromatin remodeling complex, moves DNA around histones

Front 17

17: How do boundary elements work?

Back 17

(aka insulator region)
they act like a FENCE: stops enhancer from turning on a promoter of the wrong gene.

it can also act as a boundary for CHROMATIN MODIFYING: will stop it from going further down the chromatin

Front 18

18: what is an LCR?

Back 18

LOCUS CONTROL REGION: lets you turn on access to a number of genes.

ie β Globulin: you have several different types. LCR turns on access to all the genes, which can then be individually activated.

like turning on the power to the entire building, then flipping switches in individual rooms.

Front 19

19: Are genes usually on or off?

Back 19

OFF: most need an activator to turn them on since they have a poor promoter

Front 20

20: what is the importance of SIGNAL INTEGRATION?

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there are many different activators and repressors all acting at once.

the summation of their effects together is often much more powerful than summing them individually.

you can also have one protein act as both a positive and negative signal, depending on concentration gradient.

Front 21

21: basic mechanisms of cooperative binding(4)

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1: A+B need each other to work as activators

2: A+B need to bind to a third molecule in between them to act as activators

3: A works, blocking B, but then brings in a chromatin modifier so that B can bind.

4: A binds, unwinding DNA so that B can bind.

Front 22

22: how is the HO gene controlled?

Back 22

three molecules of SWI5 bind to sites

allows CHROMATIN REMODELERS to bind

makes the SBF site available, 3 molecules SBF bind to activate gene

Front 23

23: How is the human interferon protein regulated?

Back 23

HMG proteins bend the DNA in a "U" shape so that other proteins can bind.

ENCHANCEOSOME complex forms with several proteins, all of which are needed to turn on the gene.

this complex forms in many organisms

Front 24

24: What is COMBINATORIAL CONTROL?

Back 24

one protein regulating many genes in combination with other proteins (ie CAP)

ie one gene needs activators 1, 2, 3, 4

another gene needs 3, 5, 6

Front 25

25: are most activators specific?

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some are TISSUE SPECIFIC,

most are NOT gene specific

Front 26

26: What is an example of gene control in S. Cervisae (yeast)

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mostly haploid, but when stressed will become diploid

MCM1 protein acts as an activator in certain cells, repressor in others, depending on the cell protein that it binds to.

Front 27

27: ways repressors work (4)

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COMPETITION: both trying to bind to overlapping sites

INHIBITION: both binds, but repressor occludes the activator's activating site

DIRECT REPRESSION: repressor interacts w/ mediator to stop transcription

INDIRECT REPRESSION: interacts w/ deacetylase to transform chromatin to 30nm fiber

Front 28

28: What is an example of yeast genetic repression?

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Gal1 gene:

Mig1 binds to Mig1 site.
Tup1 binds to Mig1 protein, turns off gene.

overrides UAS which promotes gene

Front 29

29: What is a second Yeast repressor?

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Gal80, occludes Gal4 activator dimer.
binds to activator when no galactose.

Front 30

30: How can we silence genes at the chromatin level?

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esp a TELOMERE:

RAP protein binds to telomere
SIR2 binds to it, then SIR 3, 4, which bind to chromatin across it

keeps telomeres condensed always.

it becomes HETEROCHROMATIN

Front 31

31: What is a long term way to silence genes?

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DNA METHYLATION: blocks activation in the long term.

proteins bind methylated DNA which blocks transcription oven more.

Front 32

32: What is another way DNA methylation could act?

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could block a BOUNDARY ELEMENT SITE: then an enhancer will act across it and turn on the wrong gene.

Front 33

33: when do you use the CHIP ASSAY?

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to amplify sections of DNA that a certain protein binds to

can do it without knowing the sequences

Front 34

34: How does the CHIP assay work?

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purify DNA (or chromatin) and shear it into fragments. can use entire chromosome

then add a binding protein, and use an antibody against it.

antibody will make a large chain from the protein, which will precipitate in solution. skim rest off, centrifuge to form a pellet.

you can remove the proteins and antibodies, and clone or PCR the DNA fragments to have pieces of all the genes that the binding protein binds to.