Mark M. Garner

The use of gel electrophoresis for quantitative studies of DNA-protein interactions is described. This rapid and simple technique involves separation of free DNA from DNA-protein complexes based on differences in their electrophoretic mobilities in polyacrylamide gels. Under favorable conditions both unbound DNA and DNA associated with protein can be quantified. This gel method is applied to the study of the E. coli lactose operon regulatory system. At ionic strengths in the physiological range, the catabolite activator protein (CAP) is shown to form a long-lived complex with the wild type lac promotor, but not with a CAP-insensitive mutant. Formation of a stable "open" or "melted-in" complex of RNA polymerase with the wild type promoter requires the participation of CAP and cyclic AMP. Further, it is demonstrated that even when pre-formed in the presence of CAP-cAMP, the polymerase-promoter open complex becomes unstable if CAP is then selectively removed.

The synthesis of a new ligand, hydrotris(methimazolyl)borate,† a soft analogue of hydrotris(pyrazolyl)borate, is reported; to demonstrate the coordination chemistry of this novel ligand, complexes of CuI and ZnII are prepared and characterised.

The nonideal properties of solutions containing high concentrations of macromolecules can result in enormous increases in the activity of the individual macromolecules. It has been proposed that molecular crowding and confinement occur in cells and are major determinants of the activity of the proteins and other intracellular macromolecules. This concept has important implications for cell volume regulation because, under crowded conditions, relatively small changes in concentration, consequent to alterations of water content, lead to large changes in macromolecular activity. This review considers several aspects of macromolecular crowding and confinement, including: 1) the physical chemical principles involved; 2) in vitro demonstrations of the effects; 3) relation to water activity; 4) estimates of the actual intracellular activity of water and macromolecules; 5) relation to osmotic regulation in various types of cells, including bacteria, red blood cells, and complex nucleated cells; and 6) the relation to inorganic ions and organic osmolytes in cells stressed by hypertonicity. We conclude that, while there is compelling evidence for important effects of molecular crowding in vitro and in red blood cells, the role of macromolecular crowding and confinement in osmotic regulation of more complex cells is an open question that deserves the extensive attention it is currently receiving.

The hydrotris(methimazolyl)borate anion (Tm), a soft analogue of the hydrotris(pyrazolyl)borate anion (Tp), has been synthesized. This novel ligand system has been designed to maintain the tripodal geometry around the boron while allowing the replacement of the three nitrogen donor atoms by three sulfur (thione) donor atoms, thus providing a complementary soft, tridentate, face capping ligand system. The two ions, Tm and Tp were compared by X-ray analysis and ab initio calculations in an attempt to explore the effects of exchanging the hard donor atoms for soft donor atoms in this type of ligand. The compound NaTm is essentially salt like with discrete anions and hydrated sodium cations. The structure of NaTp crystallised under identical conditions is observed to be an infinite ribbon containing monodentate, bridging and pendant pyrazolyl units. The co-ordination sphere of the sodium cation in NaTp is completed by two water molecules. Ab initio calculations at the Hartree–Fock level using a 6-31G* basis set on these anions and their sodium complexes suggested that while both ions are in general similar in nature, there are subtle differences which will influence their chemistry. Ab initio calculations were also used to provide a rational analysis of the formation of the two sodium salts obtained and on the analogous copper complexes further to clarify the hard and soft nature of the two ligand systems.