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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 Economize Don’t make enzymes for lactose utilization How does E. coli sense starvation for lactose? + Lactose - Lactose lactose lac expression repressed (use other carbon source) Response of E. coli to nitrogen deprivation + Nitrogen Protein synthesis Fast growth - Nitrogen Economy 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? ??? Low alanine Low cysteine … Low valine Stringent response 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 RelArelaxed (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 Amino acids: store bought RNAs: homopolymers, e.g. AAAAAAAAA... GTP and ATP: store bought or or 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? Condition AAAAA… % GTP* (p)ppGpp + Poly(A) - Poly(A) No aa’s 30.6 0.4 All aa’s 2.8 0.3 Only lysine 8.9 0.4 30.1 0.2 (AAA=lys) All but lysine 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 What happens in vivo? What does ppGpp do? Stuck!