how do bacteria respond to their environment? myxococcus starvation fruiting bodies bacillus...
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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
EconomizeDon’t make enzymes for lactose utilization
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How does E. coli sense starvation for lactose?
+ Lactose - Lactose
lac expression repressed
lactose
(use other carbon source)
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Response of E. coli to nitrogen deprivation
+ Nitrogen - Nitrogen
Protein synthesis
Fast growth
Economy
Slow growth
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Response of E. coli to nitrogen deprivationStringent response• Make less ribosomal protein• Make less ribosomal RNA• Make less transfer RNA
Problem: Multiple inputs• Deficiency in ANY amino acid = starvation
Problem: Multiple outputs• Control scores of genes
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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
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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
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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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Stringent response
???
Low alanineLow cysteine…Low valine
One small molecular signal
How does E. coli sense starvation for nitrogen?
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How does E. coli sense starvation for nitrogen?
Translocation
GTP
(requires energy)
What’s the signal?
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How does E. coli sense starvation for nitrogen?
Translocation
GDPPi
What’s the signal?
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How does E. coli sense starvation for nitrogen?
What’s the signal?
ATP
GTP
AMP
pppGpp
RelA
RelA- relaxed(no stringent response)
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Is response to N-starvation mediated through
pppGpp?1. Does pppGpp affect genes of stringent response?
2. Does uncharged tRNA pppGpp
???
Critical Questions
Ogawa & Sy (1977) J Biochem 82:947. Synthesis of guanosine polyphosphates (pppGpp and ppGpp) and its regulation by aminoacyl t-RNA
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Does uncharged tRNA pppGpp?
• In vitro system where charging of tRNA can be controlled• Assay for level of pppGpp
How to tell?
Experiment• Add uncharged tRNA to ribosomes• See if pppGpp increases
Need:
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Does uncharged tRNA pppGpp?In vitro system (~Nirenberg & Matthaei)
or or
Ribosomes:
From differential centrifugation
tRNAs: store bought
tRNA charging enzymes: From soluble fractionAmino acids: store boughtRNAs: homopolymers, e.g. AAAAAAAAA...
GTP and ATP: store bought
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Does uncharged tRNA pppGpp?Assay for level of pppGpp
pppGpp
GTP
(ATP)
GDP
ppGpp
+
+
+
+
+
Thin layer chromatography
+ amino acids - amino acids
(magic spot)
GTP* ATP
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Does uncharged tRNA pppGpp?What’s required to get pppGpp?
Condition + Poly(A) - Poly(A)
% GTP* (p)ppGpp
No aa’s
All aa’s
Only lysine
All but lysine
30.6 0.4
2.8 0.3
8.9 0.4
30.1 0.2
AAAAA…
(AAA=lys)
Maximal pppGpp depends only on absence of lysine
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Does uncharged tRNA pppGpp?Is [pppGpp] correlated with [lys-tRNA]?
Dose-response curve
Yes
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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
Stuck!
What happens in vivo?
What does ppGpp do?