Gene Expression in Prokaryotes Flashcards Preview

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0

Are genes always on?

No

1

Transcriptional control

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

2

Translational control

Governing whether ribosome sits down on the mRNA or not

3

Post-translational control

Ex: ubiquination, phosphorylation

4

Characteristics of a prokaryotic genome

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

5

Operons

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

6

Why are operons advantageous?

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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Structural genes

Code for actual enzyme

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Promoter

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

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Operator

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

10

Regulatory gene

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

11

β-galactosidase

catalyzes hydrolysis of lactose

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

O-nitrophenolate yellow, absorbs wavelength 420 nm

12

Indicator molecule

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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Model system for examining gene expression

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

14

β-galactosidase can be detected using X-gal

•  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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E.Coli experiment

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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three basic types of mutants for lac gene

lacZ-

lacY-

lacI-

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Lac Z-

gene for β-gal defective

Lactose is present, but no β-gal is made

18

Lac Y-

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

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Lac I-

• 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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LacP

Promoter

Other necessary genes are downstream

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lacZ

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

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lacY

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

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lacA

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

Gets rid of old sugars

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What happens when...
Low glucose, lactose available

lac genes strongly expressed

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

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High glucose, lactose unavailable

lac genes not expressed

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

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Low glucose, lactose unavailable

lac genes not expressed

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High glucose, lactose available

Basal level of gene expression

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

28

Repressor present, lactose absent

No transcription

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Repressor present, lactose present

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

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No repressor, lactose present/absent

Transcription occurs

31

Catabolite repression

•  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

Glucose, cAMP, CAP

•  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

When cAMP is present

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

When cAMP is absent

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

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What does glucose do?

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

Adenylyl cyclase modifies RNA base, cyclizes ATP to amp

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What happens if you have high glucose?

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

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What happens if you have low glucose?

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

38

Using partial diploids to understand gene expression

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

What mutants can be distinguished using partial diploids?

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

What's on the bacterial genome?

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

41

What is on the plasmid?

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

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Cis vs trans

•  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

Negative control vs positive control

•  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

Antibiotics

Scientists exploit what we know about bacterial translation

45

Secondary structure and the trp operon

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

Low tryptophan levels

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

High tryptophan levels

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

DNA looping

• Can be found in bacteria and humans

• AraC acts as repressor and activator

• Affected by presence or absence of arabinose

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AraC protein

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

Arabinose operon repression

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

51

Arabinose operon activation

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

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araBAD proteins produced

ara A
ara B
ara D

All have bands of equal intensity