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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?
Response of E. coli to lactose deprivation
EconomizeDon’t make enzymes for lactose utilization
How does E. coli sense starvation for lactose?
+ Lactose - Lactose
lac expression repressed
lactose
(use other carbon source)
Response of E. coli to nitrogen deprivation
+ Nitrogen - Nitrogen
Protein synthesis
Fast growth
Economy
Slow growth
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
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 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
Stringent response
???
Low alanineLow cysteine…Low valine
One small molecular signal
How does E. coli sense starvation for nitrogen?
How does E. coli sense starvation for nitrogen?
Translocation
GTP
(requires energy)
What’s the signal?
How does E. coli sense starvation for nitrogen?
Translocation
GDPPi
What’s the signal?
How does E. coli sense starvation for nitrogen?
What’s the signal?
ATP
GTP
AMP
pppGpp
RelA
RelA- relaxed(no stringent response)
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
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:
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
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
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
Does uncharged tRNA pppGpp?Is [pppGpp] correlated with [lys-tRNA]?
Dose-response curve
Yes
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?
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