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LS > Gene Expression in Prokaryotes > Flashcards

Gene Expression in Prokaryotes Flashcards

0
Q

Transcriptional control

A

Turning gene expression on and off by governing when transcription occurs and how much RNA is created

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1
Q

Are genes always on?

A

No

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2
Q

Translational control

A

Governing whether ribosome sits down on the mRNA or not

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3
Q

Post-translational control

A

Ex: ubiquination, phosphorylation

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4
Q

Characteristics of a prokaryotic genome

A

Typically genes are close to one another, no non coding sequences or introns

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5
Q

Operons

A

Genes with related function are often organized in operons

  •   Operon - an arrangement of genes in a contiguous linear array
  •   In an operon a continuous strand of mRNA carries the message for a related series of enzymes

Operons contain all genes necessary for specific bio synthetic pathway,such as converting a sugar. Different steps with different enzymes but operons contain genes for all of the enzymes. Coding sequences right next to each other. Can protein made at the same time. Coordinating the control

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6
Q

Why are operons advantageous?

A

Enzymes available right when needed. A presence of a molecule can turn on transcription.

Amounts of all enzymes coded for in the operon the same because they’re being transcribed and translated at the same time. Same intensity

  •   Genes encoding enzymes in a commonpathway can all be induced simultaneously.
  •   This type of control is called coordinate control. One mRNA expresses multiple proteins.
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7
Q

Structural genes

A

Code for actual enzyme

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8
Q

Promoter

A

where RNA polymerase binds to the DNA. Turns operon on. Recognized by recognition protein

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9
Q

Operator

A

usually adjacent to the promoter, binding site for the repressor. Important for turning on transcription

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10
Q

Regulatory gene

A

encodes the repressor protein. Can be anywhere. Does not have to be in line with the operon

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11
Q

β-galactosidase

A

catalyzes hydrolysis of lactose

When testing for activity, need an alternate. (ONPG). Gives a yellow color (chromagenic)

O-nitrophenolate yellow, absorbs wavelength 420 nm

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12
Q

Indicator molecule

A

Whenever research being done, have to have an indicator reaction that tells you if you have the protein of interest present. Test to see if the reaction occurs

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13
Q

Model system for examining gene expression

A

Jacob and Monod 1950’s &60’s

  •   Cells grown with glucose only: no β-galproduction
  •   Cells grown with glucose + lactose: no β-gal production
  •   Cells grown with lactose only: β-gal production

Only reason glucose made is that cell is glucose deficient, needs to cleave lactose

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14
Q

β-galactosidase can be detected using X-gal

A
  •   Bacterial colonies expressing β-galactosidase turn blue onagar plates containing X-gal
  •   X-gal is a substrate for β-galactosidase but is not an inducer

IPTG is added to act as the inducer

Colonies grow on plates of bacteria. X gal has beta gal linkage as well, which B galactosidase is particular to. Grow cells in presence of xgal. If bgal breaks linkage, the colony of bacteria shows a blue color. iptg is an inducer, turns on lac operon

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15
Q

E.Coli experiment

A

White colonies- normal cells.

Blue colonies are making bgal even with glucose present. But there was a mutation in operon that causes bgal to be expressed

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16
Q

three basic types of mutants for lac gene

A

lacZ-

lacY-

lacI-

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17
Q

Lac Z-

A

gene for β-gal defective

Lactose is present, but no β-gal is made

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18
Q

Lac Y-

A

No membrane protein being produced, nothing to bring lactose into the cell

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19
Q

Lac I-

A
  •  The mutants isolated mapped close to the lac operon, and this gene wasnamed the LacI gene (lac operon induction).
  •  Jacob and Monod postulated that these mutants had a defective repressor protein that normally repressed β-galactosidase synthesis. Transcription seemed to always be on

Gene for regulatory region is defective

β-gal is made with no lactose present

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20
Q

LacP

A

Promoter

Other necessary genes are downstream

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21
Q

lacZ

A

• lacZ encodes β -galactosidase, an enzyme that catalyzes the hydrolysis of lactose

22
Q

lacY

A

• lacY encodes the lactose permease, required for transport of lactose into the cell

23
Q

lacA

A

• lacA encodes a transacetylase which removes other types of β -galactosides the cell doesn’t use

Gets rid of old sugars

24
Q

What happens when…

Low glucose, lactose available

A

lac genes strongly expressed

RNA polymerase is bound tightly, and there is no repressor in the way

25
Q

High glucose, lactose unavailable

A

lac genes not expressed

RNA polymerase is bound loosely to the promoter, and also the repressor is in the way

26
Q

Low glucose, lactose unavailable

A

lac genes not expressed

27
Q

High glucose, lactose available

A

Basal level of gene expression

Even though there is no repressor, the RNA polymerase is only bound loosely to the DNA

28
Q

Repressor present, lactose absent

A

No transcription

29
Q

Repressor present, lactose present

A

Lactose binds to repressor so it cannot bind to DNA, transcription begins

30
Q

No repressor, lactose present/absent

A

Transcription occurs

31
Q

Catabolite repression

A
  •   Breakdown products of lactose are Galactose and Glucose (these are catabolites)
  •   Presence of glucose represses production of β-galactosidase. If there is already glucose present, lac operon doesn’t need to make more
32
Q

Glucose, cAMP, CAP

A
  •   Glucose metabolism is favored over lactose
  •   The level of glucose in the cell correlates with thelevel of cAMP
  •   CAP, catabolite acitvator protein, is a positive regulator which only works after binding cAMP
33
Q

When cAMP is present

A

It binds to CAP, the cAMP-CAP complex binds to DNA at the CAP site and increases binding of DNA polymerase to promoter. Transcription occurs frequently

34
Q

When cAMP is absent

A

CAP does not bind to DNA, RNA polymerase does not bind the promoter efficiently, and transcription occurs rarely

35
Q

What does glucose do?

A

Inhibits activity of enzyme adenylyl cyclase, which produces cAMP from ATP

Adenylyl cyclase modifies RNA base, cyclizes ATP to amp

36
Q

What happens if you have high glucose?

A

Inactive adenylyl cyclase, low cAMP, CAP does not bind, infrequent transcription of the lac operon

37
Q

What happens if you have low glucose?

A

Active adenylyl cyclase, high cAMP, CAP-cAMP complex binds to DNA, frequent transcription occurs

38
Q

Using partial diploids to understand gene expression

A

Haplotype- bacteria have one circular chromosome, they’re haploid.

But of mutation on operon or having a known set of genes being transcribed, can use a partial diploid. Use plasmids. Put lac operon on plasmid (naturally occurring bits of DNA).

There is one naturally occurring lac operon on the genome, and one on the plasmid introduced

39
Q

What mutants can be distinguished using partial diploids?

A

I- and Oc mutants (operator constitutive)

Hard to tell difference between these mutants. If haplotype is I- beta gal is always being made. Same with Oc

Since bacteria are haploid, a plasmid can beused to introduce a second copy of the lac operon into the cell

I- and Oc mutations behave differently in the cis versus trans arrangement (one in cis and one in trans)

40
Q

What’s on the bacterial genome?

A

lacI- which is in cis, adjacent to the operator/promoter and cannot move

41
Q

What is on the plasmid?

A

A normal lacI gene, which is in trans. Can bind to either operator.

42
Q

Cis vs trans

A

•  Trans-acting
–  any gene product that can diffuse throughthe cytoplasm and perform its function
- protein or RNA

•  Cis-dominant
–  DNA binding site which affects only the gene adjacent to it

43
Q

Negative control vs positive control

A

•  Negative control (repressor protein)
–  Operon is expressed in absence of repressor.

•  Positive control (lactose)
–  Operon is not expressed except in presence of an activator

Remember the allosteric effects that occur with the repressor& with the CAP protein. There are also proteins that can
enhance expression of a gene.

44
Q

Antibiotics

A

Scientists exploit what we know about bacterial translation

45
Q

Secondary structure and the trp operon

A

Secondary structure of RNA is important. Hairpin loops on trp operon control its expression. Transcriptional level control (prokaryotes, transcription and translation are the coupled)

46
Q

Low tryptophan levels

A

Leader sequence transcribed first, then ribosomes bind and translation begins

Slow translation of domain one due to low tryptophan levels

While the ribosome is on domain one, domain 2-3 pairing occurs and forms a hairpin which causes normal full gene transcription

47
Q

High tryptophan levels

A

Fast translation of domain one peptide

The ribosome is on domain two so 2-3 hairpin cannot form, 3-4 does instead

Hairpin makes ribosome fall off, lower intensity translation, only 10% of normal mRNAs made

48
Q

DNA looping

A
  •  Can be found in bacteria and humans
  •  AraC acts as repressor and activator
  •  Affected by presence or absence of arabinose
49
Q

AraC protein

A

C-terminal DNA binding domain and N- terminal dimerization domain

N-terminus has a arabinose binding pocket

Forms a dimer with second protein whether or not arabinose binds

50
Q

Arabinose operon repression

A

No arabinose in binding pocket. DNA loops, no transcription or translation because polymerase can’t get to the promoter

51
Q

Arabinose operon activation

A

Arabinose binds to the AraC protein and prevents it from forming a dimer, breaking the DNA loop

AraC-arabinose complexes bind to site which promotes transcription

52
Q

araBAD proteins produced

A

ara A
ara B
ara D

All have bands of equal intensity

LS (17 decks)

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