Specificity and other properties of three ribonucleases of Tetrahymena pyriformis

Abstract

Three ribonucleases, RNase I, RNase II and RNase III, were purified from the 109,000 X g supernate of detergent-treated Tetrahymena pyriformis strain W. RNases I and II act optimally at pH 5.5-6.0 and are inhibited by increasing concentrations of salts of monovalent cations. RNase III acts optimally at pH 7.5 and is activated 1.5-fold by millimolar concentrations of ZnSO4 and 5-fold by 50 mM KCl. RNases II and III are activated approximately 100% in the presence of 3 M and 5 M urea respectively. All enzymes are heat-sensitive and acid-resistant. They are endonucleases forming 2',3'-cyclic products. Their base specificity, as tested against ribosomal RNAs of known sequence, is as follows: RNase I hydrolyzes preferentially YpN and secondarily GpN bonds, RNase II is highly specific for RpN bonds, though the preparation can also hydrolyze the UpU sequence. Finally the principal targets of RNase III are YpR sequences and secondarily YpY sequences. A shorthand visualization of base specificity of nucleases in the form of right isosceles triangles is presented. The triangles are constructed by subdividing each of the two perpendicular sides in as many units as the maximum number of times the most abundant dinucleotide appears in all substrates employed and plotting the frequency of hydrolysis of each dinucleotide sequence by the enzyme under study. The proximity of each dinucleotide sequence to the hypotenuse or to one of the perpendicular sides is indicative of its susceptibility or resistance to the enzyme's action.

Several RNase T2 enzymes function in induced tRNA and rRNA turnover in the ciliate Tetrahymena

“…Subgroups of the plant RNase T2 proteins have distinct biological functions, which could be the case for ciliate Rnt2 proteins as well. Biochemical assays have suggested at least three distinct Tetrahymena RNase T2 enzyme activities (Maouri and Georgatsos, 1987; McKee and Prescott, 1991). Our work supports the physiological function of at least three T. thermophila Rnt2 proteins: Rnt2 A, B, and C. For these three paralogues, each single-gene KO produced a cellular or molecular phenotype and each double-KO combination was strongly synthetic in phenotype.…”

Ribonucleases of diverse specificities in rabbit brain nuclei

“…Thus, of three RNAses purified from mouse liver cytosol, all of which hydrolyzed poly(C) and poly(U), but not poly(A) and poly(G), two were indeed shown to be pyrimidine specific [9] but one was found to be a guanosine-specific ribonuclease [21] when allowed to act on 5s RNA. In Tetrahyrnenapyriformis one of three RNases was also shown to hydrolyze RpN bonds in 5s RNA, even though all three preferred pyrimidine polynucleotides as substrates [13]. In the present paper, Table 3 shows that, depending on the substrate used, different conclusions have to be reached for the specificity of the enzymes under investigation.…”

A guanyloribonuclease of mouse liver cytosol

“…Source of biochemicals, methodology for the preparation of RNA substrate, 3'-end-labelling of RNA, partial enzymic digests and limited alkaline hydrolysis of end-labelled RNA, electrophoresis of RNA hydrolysis products on sequencing gels and autoradiography are described in detail in an earlier paper [2]. Methods for assaying protein and RNase have also been given earlier [ 11.…”

“…Lanes 3 and 5 contained unhydrolyzed substrate (control) and a limitcd alkaline hydrolysate of the substrate respectively. Conditions for preparation of partial digests with enzymes TI and Uz as well as of limited alkaline hydrolysis are described in [2]. Conditions of preparation of partial digests with B. cereus RNase are described under Materials and Methods.…”