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367 molecular biology > pp 12 > Flashcards

pp 12 Flashcards

1
Q

What is the diauxic growth of E.coli experiment?

A

The growing E.coli feeds on glucose, when thats gone, they feed on the lactose. This can be observed by the lag phase as it needs time to express genes for lactose metabolism

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2
Q

How is lactose disaccharide hydrolyzed?

A

B-galactosidase hydrolyzes into galactose and glucose, that can be metabolized by glycolysis

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3
Q

How is B-galactosidase regulated?

A

Lac Z contains B-gal, it hydrolyzes lactose into monosaccharides and isomerizes lactose to allolactose. Lac Z, Y, A need to be present to metabolize lactose.

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4
Q

What does Lac Y contain?

A

galactoside permease that allows uptake of galactosides

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5
Q

What does Lac A contain?

A

galactoside transacetylase

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6
Q

When is the Lac operon in negative control?

A

the operon is formed when all genes are under the control of the same promoter.
Upstream is the gene with its own promoter Lac I that codes for Lac repressor. 2

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7
Q

What is Lac I?

A

Lac repressor that is a homotetrameric that has a regulatory domain that responds to presence of lactose and DNA-binding domain that binds DNA conditionally

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8
Q

What happens when there is NO inducer?

A

the lac repressor binds a DNA sequence called the operator that represses operon. This prevents RNA polymerase to transcribe the genes. When lactose is unavailable, lac operon is repressed

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9
Q

What happens when there is an inducer?

A

inducer binds to the repressor, therefore repressor can’t bind to the operator, releasing negative control. Allolactose binds the lac repressor.

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10
Q

What else can the induce the lac operon?

A

Isopropyl B-D-thiogalactoside (IPTG) it is not hydrolyzed by B-galactosidase

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11
Q

How can you tell that the binding of the lac repressor to the operator is conditional?

A

filter binding assay using labeled DNA with the operator sequence of the lac operon and repressor. When IPTG binding of repressor to operator, DNA sequence is shown to be conditional.

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12
Q

What is a trans-acting genetic element?

A

an element that can influence the expression of a 2nd gene, even if on a separate chromosomes, produce diffusible products.

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13
Q

What is a cis-acting genetic element?

A

an element that can influence the expression of a gene that is on the same chromosome.

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14
Q

What are some common regulatory mutations found in lac operon?

A

Lac I-: doesn’t code for a functional repressor
Lac Oc: operator sequence that repressor protein cannot bind

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15
Q

What can analyze binding of Lac repressor protein to the lac operator DNA sequence?

A

EMSA: observes shift in the distance traveled by DNA when bound to protein.

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16
Q

What are the three lac operators?

A

O1: adjacent to promoter
O2: upstream
O3: downstream
Co-ordinate binding of Lac repressor to O1 and either O2 or O3 leads to 1000 fold reduction in rate of transcription

17
Q

What is the positive control of the lac operon?

A

presence of lactose does not result in high expression of Lac genes if glucose is present.
Signal for low glucose is necessary to regulate expression in lac operon.

18
Q

What is the outcome of cAMP and B-galactosidase when glucose or lactose are present?

A

No glucose, no lactose: high cAMP, low B.G.
No glucose, lactose: high cAMP, high B.G.
Glucose, no lactose: low cAMP, low B.G.
Glucose, lactose: low cAMP, low B.G.

19
Q

What are the three factors involved in the position control of the lac operon

A
  1. Cyclic-AMP (cAMP)
  2. cAMP receptor protein (CRP) - protein that binds cAMP. it undergoes conformational changes allowing it to bind specific parts of DNA
  3. Binding site on DNA for CRP-cAMP
20
Q

What does the binding CRP-cAMP complex help?

A

Helps recruiting RNA polymerase to the promoter sequence

21
Q

What is Adenylate cyclase?

A
  • converts AMP to cAMP
  • its activity is inhibited by glucose
22
Q

What is the mechanism of cAMP receptor protein?

A

CRP-cAMP complex binds to CRP-binding site, with RNA polymerase
Stimulates formation of the open promoter that results in high levels of transcriptions
It positively regulates genes needed for metabolism of other sugars.

23
Q

What is the Ara operon?

A
  • Arabinose 5 carbon sugar needs araA, araB, araD genes.
  • can be positively or negatively regulated by AraC
24
Q

How is arabinose operon regulated?

A
  • expression of genes driven by araPbad promoter
  • AraC is involved in negative regulation
  • when arabinose isnt present, repressor bind to araO2 operator site and araI1
25
Q

How is the positive control of arabinose controlled?

A

arabinose is an inducer and binds AraC, causing it to bind araI1 and araI2 and unbind araO2, this opens DNA loop

26
Q

What is the gal operon?

A

For galactose
- gal repressor exerts negative control on gal operon differently from how lac repressor works as it doesn’t prevent RNA polymerase from binding promoter.
- prevents transition from closed complex to open complex, blocking the formation of elongation.

27
Q

What is the trp operon?

A

For tryptophan
- Trp operon codes for anabolic enzymes, that are tuned off by a high level of substance produced

28
Q

What is the negative control of the trp operon?

A

-negative control happens when tryptophan is present in high concentrations
-TrpR encodes aporepressor that binds tryptophan to form repressor
- binding of repressor to trpO conditionally

29
Q

What is the attenuation in the trp operon?

A
  • termination of trp operon transcription before the polymerase reaches the genes
  • Operates by causing premature termination of the operons transcript when product is abundant
30
Q

What two secondary structures available to leader-attenuator transcript?

A
  1. double hairpin secondary structure with a string of U’s
  2. less stable secondary structure with string of U’s
31
Q

What does the leader peptide have?

A
  • two trp residues in tandem
  • when trp levels are low, ribosomes stall at every trp codon
  • prevents formation of the double hairpin RNA structure (disfavours termination)
  • ribosomes moving through the hairpin favours termination
32
Q

What is the attenuation process in prokaryotes?

A
  • possible for transcriptional regulation since both happen at the same time
  • translation starts as mRNA is being elongated even before termination
33
Q

What is a Riboswitch?

A
  • RNA structure that upon binding ligand changes conformation.
  • small molecule binding elements connected to regulatory region
  • expression controlled by highly specific binding of small-molecule binding elements
  • triggers conformational change in regulatory region altering strucutre of RNA

367 molecular biology (10 decks)

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