How%20do%20bacteria%20respond%20to%20their%20environment?

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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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Response of E. coli to lactose deprivation
Economize
Dont make enzymes for lactose utilization
3
How does E. coli sense starvation for lactose?
Lactose
- Lactose
lactose
lac expression repressed
(use other carbon source)
4
Response of E. coli to nitrogen deprivation
Nitrogen
- Nitrogen
Protein synthesis
Economy
Fast growth
Slow growth
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Response of E. coli to 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

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How does E. coli sense starvation for nitrogen?
Is there a protein that binds ALL amino acids?
TheRibosome
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How does E. coli sense starvation for nitrogen?
Plenty of amino acids
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How does E. coli sense starvation for nitrogen?
Plenty of amino acids
9
How does E. coli sense starvation for nitrogen?
Plenty of amino acids
10
How does E. coli sense starvation for nitrogen?
Plenty of amino acids
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How does E. coli sense starvation for nitrogen?
Plenty of amino acids
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How does E. coli sense starvation for nitrogen?
Starvation for amino acid
Stuck!
Ribosomes can sense absence of any amino acid
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How does E. coli sense starvation for nitrogen?
Stringent response
???
Low alanine Low cysteine Low valine
One small molecular signal
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How does E. coli sense starvation for nitrogen?
Whats the signal?
Translocation
(requires energy)
GTP
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How does E. coli sense starvation for nitrogen?
Whats the signal?
Translocation
GDP
Pi
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How does E. coli sense starvation for nitrogen?
Whats the signal?
pppGpp
GTP
RelA
RelA- relaxed(no stringent response)
ATP
AMP
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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 82947. Synthesis of
guanosine polyphosphates (pppGpp
and ppGpp) and its regulation by aminoacyl t-RNA
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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

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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
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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
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Does uncharged tRNA pppGpp?
Whats required to get pppGpp?
GTP (p)ppGpp
Condition Poly(A) -
Poly(A)
No aas
30.6 0.4
All aas
2.8 0.3
AAAAA (AAAlys)
Only lysine
8.9 0.4
All but lysine
30.1 0.2
Maximal pppGpp depends only on absence of lysine
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Does uncharged tRNA pppGpp?
Yes
Is pppGpp correlated with lys-tRNA?
Dose-response curve
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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!