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 Economize Don’t make enzymes for lactose utilization

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

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

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

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

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

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

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

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

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

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

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

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

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

Does uncharged tRNA pppGpp? What’s required to get pppGpp? % GTP* (p)ppGpp Condition + Poly(A) - Poly(A) No aa’s 30.6 0.4 All aa’s 2.8 0.3 AAAAA… (AAA=lys) Only lysine 8.9 0.4 All but lysine 30.1 0.2 Maximal pppGpp depends only on absence of lysine

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

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!