Replacement of potassium chloride by potassium glutamate dramatically enhances protein-DNA interactions in vitro.
Although protein-nucleic acid interactions exhibit dramatic dependences on both ion concentration and type in vitro, large variations in intracellular ion concentrations can occur in Escherichia coli and other organisms without apparent effects on gene expression in vivo. E. coli accumulates K+ and glutamate as cytoplasmic osmolytes. The cytoplasmic K+ concentration in E. coli varies from less than 0.2 to greater than 0.9 m as a function of external osmolarity; corresponding cytoplasmic glutamate concentrations range from less than 0.03 to greater than 0.25 m. Only low levels of chloride occur in the cytoplasm of E. coli at all osmotic conditions. Since most in vitro studies have been performed in chloride salts, whereas glutamate is the more relevant physiological anion, we have measured the effects of the substitution of potassium glutamate (KGlu) for KCl on the kinetics and equilibria of a variety of site-specific protein-DNA interactions in vitro. Both the interaction of E. coli RNA polymerase with two phage lambda promoters and the interactions of various restriction enzymes with their DNA cleavage sites are enhanced by this substitution. Using the abortive initiation assay, we find a greater than 30-fold increase in the second-order rate constant for open complex formation at the lambda PR promoter and a 10-fold increase at the lambda PR' promoter, when KGlu is substituted for KCl. Replacement of KCl by KGlu does not affect the strong salt dependences of these interactions; increasing either KCl or KGlu concentrations decreases both reaction rates and extents. Substitution of glutamate for chloride does, however, shift the range of salt concentrations over which these interactions are observable to higher K+ concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)
D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005971:Glutamates / Q000494:pharmacology*; D018698:Glutamic Acid; D007700:Kinetics; D010957:Plasmids; D011189:Potassium Chloride / Q000494:pharmacology*; D011401:Promoter Regions, Genetic
D004269:DNA, Bacterial; D005971:Glutamates; D018698:Glutamic Acid; D011189:Potassium Chloride; D012321:DNA-Directed RNA Polymerases; D004262:DNA Restriction Enzymes