Compensating Effects of Opposing Changes in Putrescine (2+) and K+Concentrations onlacRepressor-lacOperator Binding:in VitroThermodynamic Analysis andin VivoRelevance

Ion concentrations (K+, Glu−) in the cytoplasm of growingEscherichia colicells increase strongly with increases in the osmolarity of a defined growth medium. Whilein vitroexperiments demonstrate that the extent of protein-nucleic acid interactions (PNAI) depends critically on salt concentration,in vivomeasurements indicate that cells maintain a relatively constant extent of PNAI independent of the osmolarity of growth. How do cells buffer PNAI against changes in the cytoplasmic environment? At high osmolarity, the increase in macromolecular crowding which accompanies the reduction in amount of cytoplasmic water in growing cells appears quantitatively sufficient to compensate for the increase in [K+]. At low osmolarity, however, changes in crowding appear to be insufficient to compensate for changes in [K+], and additional mechanisms must be involved. Here we report quantitative determinations ofin vivototal concentrations of polyamines (putrescine(2+), spermidine(3+)) as a function of osmolarity (OsM) of growth, andin vitrobinding data on the effects of putrescine concentration on a specific PNAI (lacrepressor-lacoperator) as a function of [K+]. The total concentration of putrescine in cytoplasmic water decreases at least eightfold from low osmolarity (∼64 mmol (l H2O)−1at 0.03 OsM) to high osmolarity (∼8 mmol (l H2O)−1at 1.02 OsM). Over this osmotic range the total [K+] increases from ∼0.2 mol (l H2O)−1to ∼0.8 mol (l H2O)−1. We find that the effect of putrescine concentration on the repressor-operator interactionin vitrois purely competitive and is quantitatively described by a simple competition formalism in whichlacrepressor behaves as a specific-binding oligocation (ZR=8±3). We demonstrate that this thermodynamic result is consistent with a structural analysis of the number of positively charged side-chains on two DNA binding domains of repressor which interact with the phosphodiester backbone of the operator site. Since this oligocation character of the binding surface of DNA-binding proteins appears to be general, we propose the competitive effects of putrescine and K+concentrations on the strength of specific binding are general. At low osmolarity, compensating changes in putrescine and K+concentration in response to changes in external osmolarity provide a general mechanism forE. colito vary cytoplasmic osmolarity while maintaining a constant extent of PNAI.