1. Lectures 11/12: Bacterial Adaptation Mechanisms Reading assignments in Text: Lengeler et al. 1999 Text: pages 364-368 Transcription Text: pages 437-452 Transcription mechanisms Text: pages 452-467 Transcription mechanisms Lecture 10 Text: pages 469-483 “Rapid” Enzyme control pages 123-126 ATP / NAD(P)H regulation

2. Lecture Overview Metabolism GROWTHGROWTH Differentiation Bacterial Diversity Adaptation Mechanisms Slower Change enzyme activities Make new enzymes DNARNA Protein/Enzyme Activity Rapid

3. Sensing N and Increasing GS activity Low NH 3 Sensor UT [Gln /  Keto] GS P-UMP P AT GS-AMPGS -AMP High activity No FB inhibition NRII (-) NRI ~P DNA GS glnA mRNA More GS protein ?

5. RNA polymerase / Intrinsic promoter strength  '' 2 x  Core RNA pol Catalyze RNA (Rif >) Regulators -35 -10 DNA ProteinsA/T DNA (rRNA promoters) NC NC DNA +1 RNA -35 -10 Regulator Protein A/T TTGACA TAATAT  70 “consensus” R (pol) + Pr (DNA) R-Pr (closed) Kb R-Pr (open) Kf Intrinsic promoter strength =(Kb)x(Kf)

7. Regulating intrinsic promoter strength Nitrogen regulators NRI, NRII, sigma-54 High Kb, Kf ~0 N C N C GS DNA -24 -12 -120 Enhancer UT [Gln /  Keto] P (-) NRII (-) NRI NRI~P NRII ~P Helper protein DNA bending Integration Host Helper (IHF) ATP Drives open DNA complex RNA

9. Lac operon promoter: low Kb, good Kf N C N C DNA -35 -10 TTGACA TAATAT  70 “consensus” X mismatch XX lac operon genes Activation by cAMP Receptor “CRP” cAMP -glucose +glucose RNA +Lactose Kb = 0 expected DNA -35 -10 lac operon genes -Lactose LacI = Repressor N C N C High Kb observed Kf ~ 0 “Toe-clip” Inducer +LactoseMake Inducer “quick release / quick start” N C N C

11. Why regulate? E. coli People sewer soil water Where is Lactose? Rare food, ~50% calories of milk What is Lactose? Glucose-  -1,4-Galactose Digestion? LacZ =  -galactosidase Glucose + Galactose What is the inducer? Glucose-  -1,6-Galactose “Allolactose” When does E. coli see Lactose? H. s. “LacZ” 0 birth mutants

12. Lac operon Quiz I ZYARepressorO LacZPermeaseAcetylase Allolactose IPTG = “gratuitous inducer” - IPTG +IPTG lacI- null lacI-null & F’ lacI lacI-S lacI-S & F’ lacI lacOC lacZ- null lacA- null lacZ- null &F’ lacOC lacZ lacA-null lacI-S &F’ lacOC lacZ lacA LacZ enz. LacA enz. E. coli genome (Original Jacob and Monod 1961) Wild type low High

14. Lac operon Quiz Answers I ZYARepressorO LacZPermeaseAcetylase Allolactose IPTG = “gratuitous inducer” - IPTG +IPTG lacI- null lacI-null & F’ lacI lacI-S lacI-S & F’ lacI lacOC lacZ- null lacA- null lacZ- null &F’ lacOC lacZ lacA-null lacI-S &F’ lacOC lacZ lacA LacZ enz. LacA enz. E. coli genome (Original Jacob and Monod 1961) Wild type low High low High low lacI-S dominance High zero lowHigh, but low +lactose zero low High lowHigh From F’ plasmid