title: Compensating effects of opposing changes in putrescine (2+) and K+ concentrations on lac repressor-lac operator binding: in vitro thermodynamic analysis and in vivo relevance.
abstract: Ion concentrations (K+, Glu-) in the cytoplasm of growing Escherichia coli cells increase strongly with increases in the osmolarity of a defined growth medium. While in vitro experiments demonstrate that the extent 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 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 of in vivo total concentrations of polyamines (putrescine(2+), spermidine(3+)) as a function of osmolarity (OsM) of growth, and in vitro binding data on the effects of putrescine concentration on a specific PNAI (lac repressor-lac operator) as a function of [K+]. The total concentration of putrescine in cytoplasmic water decreases at least eightfold from low osmolarity (approximately 64 mmol (l H2O)-1 at 0.03 OsM) to high osmolarity (approximately 8 mmol (l H2O)-1 at 1.02 OsM). Over this osmotic range the total [K+] increases from approximately 0.2 mol (l H2O)-1 to approximately 0.8 mol (lH2O)-1. We find that the effect of putrescine concentration on the repressor-operator interaction in vitro is purely competitive and is quantitatively described by a simple competition formalism in which lac repressor behaves a 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 for E. coli to vary cytoplasmic osmolarity while maintaining a constant extent of PNAI.
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison 53706, USA.
authors.forename: M W
authors.initials: MW
authors.lastname: Capp
authors.forename: D S
authors.initials: DS
authors.lastname: Cayley
authors.forename: W
authors.initials: W
authors.lastname: Zhang
authors.forename: H J
authors.initials: HJ
authors.lastname: Guttman
authors.forename: S E
authors.initials: SE
authors.lastname: Melcher
authors.forename: R M
authors.initials: RM
authors.lastname: Saecker
authors.forename: C F
authors.initials: CF
authors.lastname: Anderson
authors.forename: M T
authors.initials: MT
authors.lastname: Record
mesh_terms: D001483:Base Sequence; D002412:Cations; D003470:Culture Media; D003593:Cytoplasm / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry; D019077:Helix-Turn-Helix Motifs; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009875:Operator Regions, Genetic / Q000235:genetics*; D009994:Osmolar Concentration; D011188:Potassium / Q000032:analysis / Q000502:physiology*; D011485:Protein Binding; D011700:Putrescine / Q000032:analysis / Q000502:physiology*; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D013095:Spermidine / Q000032:analysis; D013816:Thermodynamics
chemical_list: D002412:Cations; D003470:Culture Media; D004269:DNA, Bacterial; D012097:Repressor Proteins; D011188:Potassium; D013095:Spermidine; D011700:Putrescine