COMPENSATING EFFECTS OF OPPOSING CHANGES IN PUTRESCINE (2-REPRESSOR LAC-OPERATOR BINDING - IN-VITRO THERMODYNAMIC ANALYSIS AND IN-VIVO RELEVANCE() AND K+ CONCENTRATIONS ON LAC)

Ion concentrations (K+, Glu-) in the cytoplasm of growing Escherichia coli cells increase strongly with increases in the osmolarity of a def ined growth medium. While in vitro experiments demonstrate that the ex tent of protein-nucleic acid interactions (PNAI) depends critically on salt concentration, in vivo measurements 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 e nvironment? At high osmolarity, the increase in macromolecular crowdin g which accompanies the reduction in amount of cytoplasmic water in gr owing cells appears quantitatively sufficient to compensate for the in crease 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 determination s of in vivo total concentrations of polyamines (putrescine(2+), sperm idine(3+)) as a function of osmolarity (OsM) of growth, and in vitro b inding data on the effects of putrescine concentration on a specific P NAI (lac repressor-lac operator) as a function of [K+]. The total conc entration of putrescine in cytoplasmic water decreases at least eightf old from low osmolarity (similar to 64 mmol (1 H2O)(-1) at 0.03 OsM) t o high osmolarity (similar to 8 mmol (1 H2O)(-1) at 1.02 OsM). Over th is osmotic range the total [K+] increases from similar to 0.2 mol (1 H 2O)(-1) to similar to 0.8 mol (1 H2O)(-1). We find that the effect of putrescine concentration on the repressor-operator interaction in vitr o is purely competitive and is quantitatively described by a simple co mpetition formalism in which lac repressor behaves as a specific-bindi ng oligocation (Z(R) = 8+/-3). We demonstrate that this thermodynamic result is consistent with a structural analysis of the number of posit ively charged side-chains on two DNA binding domains of repressor whic h interact with the phosphodiester backbone of the operator site. Sinc e this oligocation character of the binding surface of DNA-binding pro teins appears to be general, we propose the competitive effects of put rescine and K+ concentrations on the strength of specific binding are general. At low osmolarity, compensating changes in putrescine and Kconcentration in response to changes in external osmolarity provide a general mechanism for E. coli to vary cytoplasmic osmolarity while mai ntaining a constant extent of PNAI. (C) 1996 Academic Press Limited