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?