Ribonuclease II is a processive 3′ exoribonuclease in Escherichia coli. It degraded substrates with 3′-OH or 2′,3′-cyclicP ends slightly faster than those with 3′-P or 2′-P groups with a turnover number of ∼ 70 nt/s at 37°C. RNase II does not degrade DNA but the specificity for ribose was not for the cleavage bond but rather for ribo-bonds three to four nucleotides (nt) upstream, which could explain why the limit digest is a dimer. Oligonucleotides (elites) of deoxy(C) were reversible competitive inhibitors of the enzyme and indicated a strong upstream binding site (& sim; 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 residues. Compared to oligo(dC), oligos of (dA) were at least 500 times less effective inhibitors of RNase II. Using mixed oligo(dAdC) inhibitors, an obligatory 3′ to 5′ direction of binding into the catalytic site was shown.From the reaction kinetics of RNase II under different conditions 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 Vmax, while rate differences between small oligos were extreme; oligo(A)7 was degraded > 100 times faster than oligo(C)7. Ethanol, which weakens hydrophobic interactions, increased the reaction velocity of poly(C) to that of poly(A) and poly(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) was bound more strongly to a hydrophobic column than was oligo(C). Salt, which affects charge interactions, decreased the binding affinity and/or association rate of poly(C) to RNase II, had a lesser effect on poly(U), but the reactions of poly(A) were unaffected even in much higher concentrations of salt.A clue to the slower reaction velocity of poly(C) was shown when the reaction intermediates were viewed by PAGE. At lower temperatures of reaction (<25°C), there were more intense bands separated by discrete distances of ∼ 12 nt during the degradation of poly(C) by RNase II. Chase experiments showed that these stops were accounted for by dissociation of poly(C) from the enzyme. They were not seen when poly(C) was degraded at 37°C or degraded in the presence of 20% ethanol at any temperatures, nor were they seen when 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. Poly(C) is bound very strongly by ionic bonds, ∼ 15 to 27 nt from the 3′ end, to an anchor site on RNase II, while the 3′ end is pulled (threaded) through the catalytic site as the end nucleotides are cleaved off. Under conditions favoring the stacked single-strand structure, the helix is stretched to generate a progressively increasing force on the anchor site binding. After ∼ 12 nt, that binding is broken and the enzyme dissociates. With conditions that favor the random 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 strand under all these conditions. Poly(A) has a stronger helix-coil than poly(C), but binding to the anchor site is by weak hydrophobic interactions. Without strong anchor site binding, poly(A) threads through the enzyme without periodic dissociations. However, all substrates start 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 the catalytic site.
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Dec 1, 1998
Lev I Patrushev + 8
Open Access