Physiologic evaluation of the pelvic floor

In order to gain further insight into the molecular mechanism of arginine-dependent operator recognition by the hexameric Escherichia coli arginine repressor we have probed protein-DNA interactions in vitro and in vivo. We have extensively applied the chemical modification-protection and premodification-interference approach to two operators, the natural operator overlapping the P2 promoter of the carAB operon and a fully symmetrical consensus sequence. Backbone contacts were revealed by hydroxyl radical footprinting and phosphate ethylation interference. Base-specific contacts to purines and pyrimidines were revealed by methylation protection and premodification interference, KMnO4 and NH2OH.HCl-specific modification of thymine and cytosine residues, base-removal (depurination and depyrimidation), and base substitution (uracil and inosine). Additional information on the groove specificity of repressor binding was obtained by small ligand binding interference (distamycin and methyl green). In vivo, we measured the effects on the repressibility of 24 single base-pair substitutions obtained by saturation mutagenesis of half an Arg box in the carAB operator. The results of these experiments point to the conclusion that a hexameric arginine repressor molecule covers four turns of the helix, makes base-specific contacts to at least one guanine (G4 or G4') and two thymine (T3, T13', or T3', T13) residues in each one of four consecutive major grooves on one face of the helix and with four A-T/T-A base-pairs, comprising the adenine residues A9, 9', 12, 12' and the thymine residues T10, 10', 11, 11', in the two outermost minor grooves of the operator, on the very same face of the DNA molecule. The hydrophobic 5-methyl groups of four thymine residues (T3, 3', 13, 13') in each Arg box contribute to major groove-specific recognition via hydrophobic and/or van der Waals interactions. The importance of minor groove contacts was further supported by the drastic effect of distamycin binding interference. In vivo, the most pronounced drops in repressibility were occasioned by mutations at positions 10 (A-->G or C), 11 (T-->A or G) and 12 (A-->G, T or C).