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

2. Response of E. coli to lactose deprivation
Economize
Don’t make enzymes for lactose utilization

3. How does E. coli sense starvation for lactose?
lac expression repressed
(use other carbon source)

4. Response of E. coli to nitrogen deprivation
Protein synthesis
Economy
Fast growth
Slow growth

5. 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

6. How does E. coli sense starvation for nitrogen?
Is there a protein that binds ALL amino acids?
The Ribosome

7. How does E. coli sense starvation for nitrogen?
Plenty of amino acids

8. 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

11. How does E. coli sense starvation for nitrogen?
Plenty of amino acids

12. How does E. coli sense starvation for nitrogen?
Starvation for amino acid
Stuck!
Ribosomes can sense absence of any amino acid

13. How does E. coli sense starvation for nitrogen?
???
Stringent response
Low alanine
Low cysteine …
Low valine
One small molecular signal

14. How does E. coli sense starvation for nitrogen?
What’s the signal?
Translocation
(requires energy)
GTP

15. How does E. coli sense starvation for nitrogen?
What’s the signal?
Translocation Pi
GDP

16. How does E. coli sense starvation for nitrogen?
What’s the signal?
pppGpp
GTP
RelA
RelA relaxed (no stringent response)
ATP
AMP

17. 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

18. 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

19. 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

20. Does uncharged tRNA pppGpp?
Assay for level of pppGpp
GTP*
ATP +
pppGpp
Thin layer chromatography +
GTP +
(ATP) +
GDP +
ppGpp
(magic spot)
+ amino acids
- amino acids

21. Does uncharged tRNA pppGpp?
What’s required to get pppGpp?
% GTP* (p)ppGpp
Condition Poly(A) Poly(A)
No aa’s
All aa’s
AAAAA…
(AAA=lys)
Only lysine
All but lysine
Maximal pppGpp depends only on absence of lysine

22. Does uncharged tRNA pppGpp?
Is [pppGpp] correlated with [lys-tRNA]?
Yes
Dose-response curve

23. 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!