THE PROCESSIVE REACTION-MECHANISM OF RIBONUCLEASE-II

Ribonuclease II is a processive 3' exoribonuclease in Escherichia coli . It degraded substrates with 3'-OH or 2',3'-cyclicP ends slightly fas ter than those with 3'-P or 2'P groups with a turnover number of simil ar to 70 nt/s at 37 degrees C. RNase II does not degrade DNA but the s pecificity for ribose nas not for the cleavage bond but rather for rib o-bonds three to four nucleotides (nt) upstream, which could explain w hy the limit digest is a dimer. Oligonucleotides (oligos) of deoxy(C) were reversible competitive inhibitors of the enzyme and indicated a s trong upstream binding site (similar to 15 to 27 nt from the 3' end). These oligos could protect RNase II from inactivation by heat or from diethylpyrocarbonate, an agent that preferentially reacts with His res idues. Compared to oligo(dC), oligos of (dA) were at least 500 times l ess effective inhibitors of RNase II. Using mixed oligo(dAdC) inhibito rs, an obligatory 3' to 5' direction of binding into the catalytic sit e was shown. From the reaction kinetics of RNase II under different co nditions it was concluded that the enzyme recognition differs for poly (A), poly(C) and poly(U). Poly(C) was degraded more slowly than poly(A ) or poly(U) with a 3.5 times slower V-max, while rate differences bet ween small oligos were extreme; oligo(A)(7) was degraded >100 times fa ster than oligo(C)(7). Ethanol, which weakens hydrophobic interactions , increased the reaction velocity of poly(C) to that of poly(A) and po ly(U). It had no effect on the reaction velocities of poly(A) or poly( U), but decreased the binding of poly(A) markedly. Oligo(A) ws bound m ore strongly to a hydrophobic column than was oligo(C). Salt, which af fects charge interactions, decreased the binding affinity and/or assoc iation rate of poly(C) to RNase II, has a lesser effect on poly(U), bu t the reactions of poly(A) were unaffected even in much higher concent rations of salt. A clue to the slower reaction velocity of poly(C) was shown when the reaction intermediates were viewed by PAGE. At lower t emperatures of reaction (<25 degrees C), there were more intense bands separated by discrete distances of similar to 12 nt during the degrad ation of poly(C) by RNase II. Chase experiments showed that these stop s were accounted for by dissociation of poly(C) from the enzyme. They were not seen when poly(C) was degraded at 37 degrees C or degraded in the presence of 20% ethanol at any temperatures, nor were they seen w hen poly(A) or poly(U) was degraded even at low temperatures. However, all substrates showed dissociation when the oligo became less than 10 to 15 nt. A model was proposed to account for these observations. Pol y(C) is bound very strongly by ionic bonds, similar to 15 to 27 nt fro m the 3' end, to an anchor site on RNase II, while the 3' end is pulle d (threaded) through the catalytic site as the end nucleotides are cle aved off. Under conditions favoring the stacked single-strand structur e, the helix is stretched to generate a progressively increasing force on the anchor site binding. After similar to 12 nt, that binding is b roken and the enzyme dissociates. With conditions that favor the rando m coil (higher temperature or ethanol) these stops are not seen. This is the case with poly(U), which tends to be a randomly coiled single s trand under all these conditions. Poly(A) has a stronger helix-coil th an poly(C), but binding to the anchor site is by weak hydrophobic inte ractions. Without strong anchor site binding, poly(A) threads through the enzyme without periodic dissociations. However, all substrates sta rt dissociating with each cleavage when they become so small that the anchor site cannot be filled by nucleotides. In this model the energy for progression is provided by the pulling force on the substrate at t he catalytic site.