INTERACTION OF THE BASIC PROTRUSION OF ESCHERICHIA-COLI RIBONUCLEASE HI WITH ITS SUBSTRATE

In order to determine the actual distance between the active site and the substrate binding site, termed the basic protrusion, of Escherichi a coli ribonuclease HI, synthetic oligonucleotide duplexes with gradua lly extended overhangs were used, in which the enzymatic cleavage was restricted to a single site with 2'-O-methylnucleosides. The affinity of the enzyme for each substrate was determined by kinetic analysis. I t was found that the affinity increased markedly when one nucleotide w as attached to the 3' end of the DNA strand of the nine-base-pair hybr id duplex and then increased slightly as the DNA strand was extended f urther, whereas elongation of the strand in the other direction caused no change. When a mutant enzyme, in which three lysine residues in th e basic protrusion were altered to alanine, was used, no increase in t he k(cat)/K-m value was observed. The results indicate that, for the p roductive binding, the axis from the 3' to the 5' end of the RNA stran d of the substrate duplex must be oriented in agreement with the direc tion from the active site to the basic protrusion of the enzyme. The d istance between the active site and the basic protrusion in the enzyme -substrate complex was shorter than that anticipated in modeling studi es. A dynamic structure refinement, referred to as the normal mode ana lysis, was carried out in order to simulate the fluctuations of the ba sic protrusion. (C) 1996 Academic Press Limited