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3130 > Chapter 7 > Flashcards

Chapter 7 Flashcards

1
Q

Define an operon

A

A group of functionally related genes, regulated by a single promoter/operator region.

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2
Q

T/F: When a group of genes are controlled by an operon, each gene is transcribed in the same amount each time the operon is activated.

A

False. Each gene can still be individually controlled to create more or less of each gene product.

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3
Q

T/F: The Lac operon controls the synthesis of genes responsible for the metabolism of lactose

A

True

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4
Q

What genes are controlled by the lac operon?

A

Lac Y, Lac Z and Lac A

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5
Q

What are the purposes of the lac genes?

A

Lac Z - transports lactose into cells
Lac Y - Cleaves lactose into galactose and glucose
Lac A - Unclear

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6
Q

What is the protein product of Lac Z?

A

Galactoside permease

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7
Q

What is the protein product of Lac Y?

A

β-galactosidase

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8
Q

What is the protein product of Lac A?

A

Galactoside transacetylase

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9
Q

How many RNA strands are produced from activation of the lac operon

A

One

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10
Q

Define cistron

A

A gene

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11
Q

Define polycistronic

A

Multiple genes

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12
Q

T/F: There is only one ribosomal binding site on a polycistronic RNA.

A

False, each gene has its own ribosomal binding site and its translation can therefore be controlled independent of the other genes.

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13
Q

What is negative control?

A

Control where genes are not transcribed due to the presence of a repressor.

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14
Q

What gene codes for the lac operon repressor?

A

LacI

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15
Q

Is the lac operon positively or negatively controlled?

A

Negativelly

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16
Q

T/F: The LacI repressor is a heterodimer of four polypeptides

A

False, it is a homodimer of four identical polypeptides.

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17
Q

T/F: The LacI protein promotes transcription of the Lac operon

A

False, it represses transcription.

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18
Q

T/F: The lac repressor binds to DNA to prevent RNA polymerase from binding to the promoter.

A

True

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19
Q

T/F: The lac operon is repressed when lactose is available

A

False (for this class). It is repressed if lactose is NOT available.

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20
Q

What causes the LacI gene to dissociate from DNA?

A

An inducer molecule binds to the repressor, causing a conformational change which makes it fall off.

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21
Q

Define allosteric protein

A

A protein in which binding of a molecule to one site changes the conformation of a remote site and alters its interaction with a second molecule.

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22
Q

What is the inducer for the lac operon?

A

Allolactose

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23
Q

How is allolactose generated by a cell?

A

β-galactosidase rearranges the β-1,4 bond in lactose to a β-1,6 bond, creating allolactose.

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24
Q

What function(s) does β-galactosidase perform?

A

It can cleave the β-1,4 glycosidic bond in lactose, or rearrange it to form a β-1,6 glycosidic bond, creating allolactose.

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25
Q

How can β-galactosidase rearrange lactose if the operon containing this enzyme is repressed?

A

There is always a small, “leaky” amount of gene transcription, so a small amount of β-galactosidase is always available.

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26
Q

What type of experiment was used to show that a repressor binds to the lac operator?

A

A filter-binding assay

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27
Q

What type of experiment was used to show that an inducer prevents binding of the lac operon repressor?

A

A filter-binding assay

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28
Q

What is IPTG?

A

A synthetic inducer that can be used to mimic allolactose.

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29
Q

What were the results of the assay used to show that the lac operon is controlled by a repressor?

A

When an artificial inducer (IPTG) was added, the percentage of repressor bound to DNA became very low, while if there was no IPTG, binding reached saturation.

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30
Q

Describe the experiment used to show that repressor prevents polymerase binding to the lac promoter.

A

RNA polymerase and lac promoter were allowed to form a complex.

They measured the rate of transcript synthesis, either alone or in the presence of heparin, or in the presence of the lac repressor.

Transcription was measured using a UTP analog, fluorescently tagged on the gamma-phosphate. UMP incorporation into RNA thus released a fluorescent pyrophosphate.

(The conclusion of this assay is flawed)

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31
Q

Why did researchers conclude that repressor prevents polymerase binding to DNA?

A

Because the levels of transcription when repressor was added to a DNA-repressor complex were similar to those of when heparin was added, and we know heparin prevents binding (however, this isn’t really supported and correlation does not equal causation).

32
Q

How many operators does the lac operon have?

A

Three

33
Q

What are auxiliary operators?

A

The minor operators that function in addition to the main operator (which is found close to the transcription start site).

One auxiliary operator is upstream of the primary operator, and one is downstream.

Removal of one auxiliary operator depresses repression, and removal of both can depress it as much as 50X.

34
Q

How are the monomers in the lac repressor arranged?

A

Two connected pairs of dimers to make a homotetramer.

35
Q

T/F: A lac repressor can only bind to one operator.

A

False. The dimers that make the tetramer can bind independently to separate lac operators.

36
Q

T/F: Removing both auxiliary operators only causes a slight decrease in repression.

A

False. Repression decreases 50X.

37
Q

What molecule is necessary for positive control of the lac operon?

A

cAMP + CAP

38
Q

What is cAMP?

A

Cyclic AMP. It is involved in positive control of the lac operon.

39
Q

Why does cAMP increase when glucose levels are low?

A

Because production of the enzyme that makes cAMP increases when glucose levels are low (kinda a non-answer imo)

40
Q

How do we know cAMP is involved in positive regulation of the lac operon?

A

cAMP added to bacteria will cause increased transcription of the lac genes, even when glucose is present.

41
Q

Can cAMP positively control the lac operon by itself?

A

No, it needs to form a complex with CAP (catabolite activator protein).

42
Q

What does CAP stand for?

A

Catabolite Activator Protein

43
Q

How does the CAP-cAMP complex influence transcription?

A

It helps the RNA polymerase bind to the promoter and form the OPC.

44
Q

Where is the CAP-cAMP activator site?

A

Just upstream (5’) of the promoter.

45
Q

Describe the experiment used to show whether the CAP-cAMP complex helped open the promoter.

A

First, RNA was allowed to bind to polymerase in the presence or absence of CAP-cAMP.

Next, rifampicin and NTPs were added simultaneously.

Then they measured whether transcription occurred. If it did, that meant the bubble was already open because transcription would initiate before rifampicin had time to block it. This is what occurred.

Hudak doesn’t think this is the best setup, but didn’t offer an alternative other than that she thought rifampicin and NTPs were too close in size.

46
Q

How do we know CAP-cAMP binds upstream of the polymerase binding site?

A

1) mutations to the CAP-cAMP binding site prevent stimulation by CAP-cAMP, but a low level of transcription still occurs.

2) DMS footprinting analysis

47
Q

Describe DMS footprinting

A

1) End-label one strand of DNA and allow protein to bind.

2) Mildly methylate (preferably one methylation per strand) by adding DMS.

3) Remove the protein.

4) Treat DNA with piperidine which removes the methylated purines and cleaves DNA at apurinic sites (breaks up into fragments cut at methyl sites)

5) Electrophorese on a urea-acrylimide gel.

6) Where fragments are missing as opposed to the control is where the protein bound. Thicker bands indicate places where the G was more exposed due to changes in DNA shape.

48
Q

What information does DMS footprinting provide?

A

Both where the protein bound to the DNA and whether it changes the shape of the DNA.

49
Q

What is the advantage of using DMS over DNA nuclease?

A

DMS is much smaller and can fit in spaces DNA nuclease cannot, providing more detailed information about protein shape.

50
Q

T/F: The lac operon’s -35 is highly conserved

A

False. It bears little similarity to the conserved sequence and therefore the polymerase only binds weakly to it.

51
Q

Does the lac operon have a strong promoter region and what are the consequences of this?

A

No, it’s fairly weak. If it were strong then the operon would be always on and lac genes would always be transcribed. This would be energetically inefficient for the cell.

52
Q

What are the two steps of recruitment to the promoter by CAP-cAMP?

A

1) Formation of the CPC
2) Conversion of the CPC to the OPC.

53
Q

T/F: The CAP-cAMP directly increases the rate of conversion from the CPC to the OPC.

A

False. It only increases the rate of formation of the CPC. But by doing so it increases the probability of the OPC being formed, indirectly increasing the rate of OPC.

54
Q

How does CAP-cAMP binding to the activator site help to recruit RNA polymerase to the promoter?

A

1) CAP and polymerase touch as they bind to their DNA sites, and binding is co-operative between the two proteins.
2) CAP-cAMP binding causes the DNA to bend.

55
Q

Name the five types of evidence we have that CAP and polymerase touch as they bind to their DNA sites.

A

1) CAP and polymerase co-sediment on gradient ultracentrifugation in the presence of cAMP.
2) CAP and polymerase can be cross-linked covalently when both are bound to their DNA sites.
3) DNase footprinting assays.
4) Mutations to the alpha-CTD of polymerase (thought to be the site to interact with the activation region (ARI) of CAP prevents actiavtion by CAP-cAMP.
5) X-ray crystallography shows that the ARI site touches alpha-CTD.

56
Q

What is the ARI?

A

Activation Region

57
Q

How do we know CAP-cAMP bends DNA?

A

Linear DNA fragments were prepared from a plasmid, all with the CAP-cAMP location in different places on the fragment. CAP-cAMP was then bound to the fragments and electrophoresed. If CAP-cAMP binding bent the DNA the fragments should migrate differently, which is what was seen.

58
Q

Why do proteins bend DNA?

A

To either improve it’s own ability to bind to DNA, or to assist another protein in binding to DNA.

59
Q

What type of genes does the trp operon encode for and what do they do?

A

Anabolic enzymes that synthesize tryptophan.

60
Q

What type of operon is the trp operon?

A

Anabolic

61
Q

What is the precursor to tryptophan?

A

Chorismic acid.

62
Q

When is the trp operon repressed?

A

When W levels are high.

63
Q

Where is the trp operator?

A

Entirely within the trp promoter.

64
Q

What is an aporepressor?

A

A protein which is a repressor, but in its inactive form because it does not have its corepressor.

65
Q

When is the trp operon active?

A

When there is not enough W in the cell to activate the aporepressor.

66
Q

T/F: Trp operon repression is strong and results in 700X less transcription.

A

False. Only 70X repression. Requires an attenuator to get to 700X.

67
Q

T/F: The first gene of the trp operon is trpA

A

False, it is trpE

68
Q

What is the order of regions on the trp operon?

A

trpO, P (binding site), trpL (leader), attenuator, trp E.

69
Q

Describe trpL

A

This is the leader sequence. It is 14 aa long. It starts with a Met and ends with Ser.

70
Q

Which product of initial trp transcription is more stable, the 2-hairpin or the 1-hairpin?

A

2-hairpin

71
Q

Describe the process that leads to the termination of transcription in the trp operon.

A

When tryptophan is scarce, the ribosomes translating the nascent RNA will need to pause when they reach the double UGG sequence that calls for tryptophan. This prevents elements 1 and 2 from forming a hairpin, and elements 2 and 3 will form a hairpin instead. No hairpin then forms between 3 and 4, leading to the polymerase transcribing through the AT-region and on to the rest of the operon.

72
Q

Which helices make up the platform of the trp aporepressor?

A

A, B, C, and F

73
Q

Which helices of the trp aporepressor lie next to the DNA?

A

D and E

74
Q

How does W modify the aporepressor?

A

It causes helix E to shift, shifting it so it points directly into the major groove of DNA (90 degrees instead of ~75).

75
Q

Why does TRP binding to the aporepressor cause increased binding?

A

Helix E moves into a position that provides better contact with the major groove of DNA.

76
Q
A

3130 (5 decks)

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