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How do bacteria respond to their environment?. Myxococcus Starvation Fruiting bodies. Bacillus Starvation Spores. Anabaena Starvation Heterocysts. How is starvation sensed and interpreted?.

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How do bacteria respond to their environment
How do bacteria respond to their environment?

  • MyxococcusStarvation Fruiting bodies

  • BacillusStarvation Spores

  • AnabaenaStarvation Heterocysts

How is starvation sensed and interpreted?


Response of e coli to lactose deprivation
Response of E. colito lactose deprivation

Economize

Don’t make enzymes for lactose utilization


How does e coli sense starvation for lactose
How does E. coli sense starvation for lactose?

+ Lactose

- Lactose

lactose

lac expression repressed

(use other carbon source)


Response of e coli to nitrogen deprivation
Response of E. colito nitrogen deprivation

+ Nitrogen

- Nitrogen

Protein synthesis

Economy

Fast growth

Slow growth


Response of e coli to nitrogen deprivation1
Response of E. colito nitrogen deprivation

  • Stringent response

  • Make less ribosomal protein

  • Make less ribosomal RNA

  • Make less transfer RNA

  • Problem: Multiple outputs

  • Control scores of genes

  • Problem: Multiple inputs

  • Deficiency in ANY amino acid = starvation


How does e coli sense starvation for nitrogen
How does E. coli sense starvation for nitrogen?

Is there a protein that binds ALL amino acids?

TheRibosome


How does e coli sense starvation for nitrogen1
How does E. coli sense starvation for nitrogen?

Plenty of amino acids


How does E. coli sense starvation for nitrogen?

Plenty of amino acids


How does E. coli sense starvation for nitrogen?

Plenty of amino acids


How does E. coli sense starvation for nitrogen?

Plenty of amino acids


How does E. coli sense starvation for nitrogen?

Plenty of amino acids


How does E. coli sense starvation for nitrogen?

Starvation for amino acid

Stuck!

Ribosomes can sense absence of any amino acid


How does e coli sense starvation for nitrogen2
How does E. coli sense starvation for nitrogen?

???

Stringent response

Low alanine

Low cysteine

Low valine

One small molecular signal


How does E. coli sense starvation for nitrogen?

What’s the signal?

Translocation

(requires energy)

GTP


How does E. coli sense starvation for nitrogen?

What’s the signal?

Translocation

Pi

GDP


How does E. coli sense starvation for nitrogen?

What’s the signal?

pppGpp

GTP

RelA

RelA- relaxed(no stringent response)

ATP

AMP


Is response to n starvation mediated through pppgpp
Is response to N-starvation mediated through pppGpp?

Critical Questions

1. Does pppGpp affect genes of stringent response?

2.Does uncharged tRNA pppGpp

???

Ogawa & Sy (1977) J Biochem 82:947. Synthesis of guanosine polyphosphates (pppGpp and ppGpp) and its regulation by aminoacyl t-RNA


Does uncharged trna pppgpp
Does uncharged tRNA pppGpp?

How to tell?

  • Experiment

  • Add uncharged tRNA to ribosomes

  • See if pppGpp increases

Need:

  • In vitro system where charging of tRNA can be controlled

  • Assay for level of pppGpp


Does uncharged trna pppgpp1
Does uncharged tRNA pppGpp?

In vitro system (~Nirenberg & Matthaei)

Ribosomes:

From differential centrifugation

tRNAs:store bought

tRNA charging enzymes:

From soluble fraction

or

or

Amino acids: store bought

RNAs: homopolymers, e.g. AAAAAAAAA...

GTP and ATP: store bought


Does uncharged trna pppgpp2
Does uncharged tRNA pppGpp?

Assay for level of pppGpp

GTP*

ATP

+

pppGpp

GTP

Thin layer chromatography

+

(ATP)

+

GDP

+

ppGpp

+

(magic spot)

+ amino acids

- amino acids


Does uncharged trna pppgpp3
Does uncharged tRNA pppGpp?

What’s required to get pppGpp?

% GTP* (p)ppGpp

Condition + Poly(A) - Poly(A)

No aa’s

30.6 0.4

All aa’s

2.8 0.3

AAAAA…

(AAA=lys)

8.9 0.4

Only lysine

All but lysine

30.1 0.2

Maximal pppGpp depends only on absence of lysine


Does uncharged trna pppgpp4
Does uncharged tRNA pppGpp?

Yes

Is [pppGpp] correlated with [lys-tRNA]?

Dose-response curve


Does uncharged trna pppgpp5
Does uncharged tRNA pppGpp?

  • In vitro, Lys induces pppGpp

  • In vitro, Lys induces Lys-tRNA

  • In vitro, Lys-tRNA correlated with pppGpp

What happens in vivo?

What does ppGpp do?

Stuck!


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