Specificity and other properties of three ribonucleases of Tetrahymena pyriformis

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.     

Hydrogen exchange and the proteolytic degradation of ribonuclease A. The position of the centers of the high-temperature splitting by proteases and the structure of denatured molecules

The information on the high-temperature proteolytic degradation of RNase A has been analyzed. It has been shown that a few peptide bonds primarily splitted by trypsin, chymotrypsin and thermolysin are localized only in the N-terminal part of structural domain II of the native molecule. The same peptide bonds are splitted by proteases with the highest rate upon the denaturation in the presense of trifluoroethanol or the renaturation from concentrated urea solutions and after the desorganization of the native structure by the reduction of all four S-S bonds of RNase A. According to the data on hydrogen exchange in the native RNase A molecule, the dynamic stability of the tertiary structure of domain II is lower than that of domain I because of the lesser amount of the internal bulky nonpolar residues Val, Ile, and Phe. For the same reason, this part of the molecule in different nonnative forms of RNase A is less compact and more flexible and is splitted with the highest rate in the segment 31-39 enriched by long cationic residues Lys and Arg. A common feature of the conformation of the flexible disordered backbone of all RNase A nonnative structures considered is the predominance of short PPII helices, which provides a high rate of the restoration of the native secondary and tertiary structures upon renaturation or self-organization and global fluctuations of the native structure revealed by the hydrogen exchange and proteolytic degradation.     

Effect of neurectomy on nuclease activity in skeletal muscles of rats

Author followed up the activity of the three enzymes involved in the catabolism of nucleic acids--acid deoxyribonulease (DNase II), alkaline ribonuclease (RNase I), and acid ribonuclease (RNase II)--in the denervated gastrocnemius and soleus muscles of rats for 28 postoperative days. The activity of both acid nucleases increased in both types of denervated muscles, compared with the respective controls. Up to the 14th postoperative day, the activity excess of both acid nucleases was more significant in the m. gastrocnemius than in the m. soleus. The RNase I ran below the control activity during the whole period in the m. soleus and up to the 14th day in the m. gastrocnemius. The role of nucleases and nuclease inhibitors in the changes of nucleic acid catabolism in neurogenic muscular atrophies is discussed.     

RNases in ColE1 DNA metabolism

ColEl DNA replication is initiated by RNA II and inhibited by RNA I. Control of the replication occurs through the interaction between RNA I and RNA II. Therefore, RNases involved in the metabolism of RNA I and RNA II are expected to play a key role in the control of the ColEl plasmid replication. RNase H, RNase E, RNase III, RNase P, and polynucleotide phosphorylase carry out the many specific reactions of the RNA metabolism.     

The amino acid sequence of ribonuclease N1, a guanine-specific ribonuclease from the fungus Neurospora crassa

The complete amino acid sequence of ribonuclease N1 (RNase N1), a guanine-specific ribonuclease from a fungus, Neurospora crassa, was determined by conventional protein sequencing, using peptide fragments obtained by tryptic digestion of cyanogen bromide-treated RNase N1 and by Staphylococcus aureus V8 protease digestion of heat-denatured RNase N1. The results showed that the protein is composed of a single polypeptide chain of 104 amino acid residues cross-linked by two disulfide bonds and has a molecular weight of 11,174: (sequence; see text) (Disulfide bonds: C2-C10, C6-C103) The amino acid sequence was homologous with those of RNase T1 (65% identity) and related microbial RNases.     

Human liver acid phosphatases.

Human liver contains three chromatographically distinct forms of non-specific acid phosphatase (EC 3.1.3.2). Acid phosphatases I, II and III have molecular weights of greater than 200 000, of 107 000, and of 13 400, respectively. Following partial purification, isoenzyme II was obtained as a single activity band, as assessed by activity staining with p-nitrophenyl phosphate and alpha-naphthyl phosphate on polyacrylamide gels run at several pH values. With 50mM p-nitrophenyl phosphate as a substrate, enzymes II and III exhibit plateaus of activity over the pH range 3 - 5 and 3.5 - 6, respectively.Acid phosphatase II is not significantly inhibited by 0.5% formaldehyde. The activity of human liver acid phosphatase II and of human prostatic acid phosphatase towards several substrates is compared. The liver enzyme, is marked contrast to the prostatic enzyme, does not hydrolyze O-phosphoryl choline.     

Multicompartmental distribution of ribonuclease II in mouse liver

Thirty percent of RNase II (EC 3.1.27.5) is present in the cytosol of mouse liver where it exists in an inactive complex with a protein inhibitor. The remaining 70% of RNase II is active, soluble enzyme unassociated with inhibitor and is distributed in a ratio of 1.3 to 1 between the lumen of reticular elements and the interior of heavy particles. Although heavy particle RNase II resembles acid hydrolases in centrifugal behavior, in other tests including density shift experiments the resemblance is incomplete. In experiments employing lysis induced by L-amino acid methyl esters, RNase II activity is much more latent than the activity of the lysosomal marker, acid RNase. It is postulated that the heavy particle component of RNase II is contained in a secretory vesicle rather than in classic lysosomes.     

Purification and properties of three cytosolic ribonucleases of mouse liver

The ribonucleolytic activity of mouse liver cytosol is due to at least three different enzymes, whose purification is reported. Two of these enzymes, an alkaline and a neutral RNase, have specificities practically identical with that of pancreatic RNase. The third enzyme, an acid RNase, is highly specific for NpU bonds where N is A, G or C and also cleaves ApG bonds provided they are part of a GpApG sequence and preferentially a GpApGpA repeat.     

Purification and characterization of ribonuclease M and mRNA degradation in Escherichia coli

A previously unreported endoribonuclease has been identified in Escherichia coli, which has a preference for hydrolysis of pyrimidine-adenosine (Pyd-Ado) bonds in RNA. It was purified about 7000-fold to give a single band after SDS/polyacrylamide gel electrophoresis; the eluted protein gave the same RNase specificity. The sizes of the native and denatured enzymes agreed suggesting that the enzyme exists as a monomer of approximately 26 kDa. It is called RNase M. The only other reported broadly specific endoribonuclease in E. coli is RNase I, a periplasmic enzyme. Based on differences in charge, heat stability and substrate specificity, it was clear that RNase M is not RNase I. The specificity of RNase M was remarkably similar to that of pancreatic RNase A even though the two enzymes differ in charge characteristics and size. Earlier studies had shown that mRNA from the lactose operon of E. coli is hydrolyzed in vivo primarily between Pyd-Ado bonds [Cannistraro et al. (1986) J. Mol. Biol. 192, 257-274] We propose that this major RNase activity accounts for these cleavages observed in vivo and that it is the endonuclease for mRNA degradation in E. coli.     

Toxic Metabolites of an Unidentified Filamentous Fungus Isolated from Zinnia Leaves

Two ribonucleases (RNases) designated RNase I and RNase II were found in Euphausia superba and isolated by (NH₄)₂SO₄ fractionation, 2 cycles of CM-cellulose chromatography and gel filtration on Sephadex G-100. This procedure resulted in a 2,116-fold purification of RNase I and a 130-fold purification of RNase II. The molecular weight of both purified enzymes was estimated by gel filtration to be 31,500. The isoelectric points were 6.0 (RNase I) and 7.0 (RNase II). Each enzyme hydrolyzed poly A-U, poly U but did not degrade poly G, poly C and DNA. Both enzymes were classified as endonuclease from the hydrolysis product of yeast RNA and poly A. The enzymes were located mainly in the cardiac and pyloric portion of the stomach.     

Adenosine diphosphate ribosylation of histone H1 by purified calf thymus polyadenosine diphosphate ribose polymerase

The mechanism of poly ADPR synthesis and the transfer of poly ADPR to histone H1 molecule by electrophoretically homogenous calf thymus poly ADPR polymerase containing DNA was examined. 1) An acid insoluble radioactive complex (I) was obtained after incubation of purified enzyme with [3H] NAD. The stability of (I) was examined by SDS-polyacrylamide gel electrophoresis. The complex (I) was stable against acid, SDS, urea, DNase and RNase, but labile against pronase, trypsin, alkali and snake venom phosphodiesterase treatment. The molecular weight of (I) was about 130 000 daltons estimated by SDS-gel electrophoresis. The radioactive products of successive alkali, venom phosphodiesterase and Pronase hydrolysis of (I) were PR-AMP and AMP. The mean chain length of poly ADPR of (I) was 20--30. These results suggest that the complex (I) is poly ADP-ribosylated poly ADPR polymerase. 2) Besides (I), a second radioactive peak (II) was observed when acid insoluble products obtained from an incubation mixture containing purified poly ADPR polymerase, [3H] NAD and purified histone H1 were analyzed on SDS-polyacrylamide gel electrophoresis. The molecular weight of (II) was estimated to be about 23 000 daltons. The complex (II) is eluted like histone H1 on CM-cellulose columns and hydrolyzed by alkali, trypsin and snake venom phosphodiesterase but not by DNase, or RNase. The comples (II) was extracted selectively by 5 per cent perchloric acid or 5 per cent trichloroacetic acid from mixture of (I) and (II). The mean chain length of poly ADPR of complex (II) and 5--20; these results suggest that the complex (II) is poly ADP-ribosylated histone H1. 3) Results 1) and 2) indicate that purified DNA containing, thus DNA independent, poly ADPR polymerase catalyzes two different reactions, the ADPR transfer onto the enzyme itself and onto histone H1 and the elongation of ADPR chains. Dimeric forms of ADP-ribosylated histone H1 was not observed. Free poly ADPR was observed only when very small quantities of enzyme were used for incubation.     

Expression of bovine pancreatic ribonuclease A coded by a synthetic gene in Bacillus subtilis

Two different hybrid genes were constructed which fuse the Bacillus amyloliquefaciens alkaline protease gene (apr[BamP]) promoter and signal peptide coding region to a synthetic bpr gene coding for the mature bovine pancreatic RNase A. The first gene fusion (apr-bpr1) contained the apr[BamP] signal peptide coding region fused to mature bpr through a linker coded 3-amino acid region and retained the signal processing site ala-ala of the alkaline protease. The second fusion (apr-bpr2) joined the end of the apr[BamP] signal peptide coding sequence to the mature bpr resulting in a hybrid signal processing site ala-lys. B. subtilis strains harboring these gene fusions secreted bovine pancreatic RNase A into the growth medium. Cleavage at the hybrid signal processing site ala-lys resulted in the secretion of bovine pancreatic RNase A from B. subtilis which had an N-terminal amino acid sequence that was identical to the native RNase A. Bovine pancreatic RNase A contains four disulfide bonds and the proper formation of these bonds is required for activity. RNase activity could be detected in the culture supernatants of strains carrying the apr-bpr gene fusions, which suggests that the proper disulfide bonds have formed spontaneously.     

Expression, purification, and crystallization of natural and selenomethionyl recombinant ribonuclease H from Escherichia coli

Ribonuclease H (RNase H) from Escherichia coli is an endonuclease that specifically degrades the RNAs of RNA:DNA hybrids. The enzyme is a single polypeptide chain of 155 amino acid residues, of which 4 are methionines. To solve the crystallographic three-dimensional structure of E. coli RNase H by the multi-wavelength anomalous diffraction technique, we have constructed methionine auxotrophic strains of E. coli that overexpress selenomethionyl RNase H. MIC88 yields about 10 mg of selenomethionyl RNase H per liter of culture, which is comparable to the overexpression of the natural recombinant protein. We have purified both proteins to homogeneity and crystallized them isomorphously in the presence of sulfate. These are Type I crystals of space group P2(1)2(1)2(1) with the cell parameters a = 41.8 A, b = 86.4 A, c = 36.4 A, one monomer per asymmetric unit, and approximately 36% (v/v) solvent. Crystals of both proteins diffract to beyond 2-A Bragg spacings and are relatively durable in an x-ray beam. On replacement of sulfate with NaCl, crystals of natural RNase H grow as Type I' (very similar to Type I) at pH between 7.0 and 8.0; at pH 8.8, crystals of Type II are obtained in space group P2(1)2(1)2(1) with a = 44.3 A, b = 87.3 A, and c = 35.7 A. Type II crystals can be converted to Type I by soaking in phosphate buffer. RNase H crystals of Type II have also been reported by Kanaya et al. (Kanaya, S., Kohara, A., Miyakawa, M., Matsuzaki, T., Morikawa, K., and Ikehara, M. (1989) J. Biol. Chem. 264, 11546-11549).     

Amino acid sequence and S-S bonds of Penicillium brevicompactum guanyl-specific ribonuclease

The primary structure of Penicillium brevicompactum guanyl-specific RNase was determined. The enzyme consists of 102 amino acid residues, Mr 10801. The 4 cysteine residues of the RNase are linked in pairs by disulfide bonds: Cys2-Cys10, Cys6-Cys101. P. brevicompactum RNase structure is similar to RNase T1; the degree of homology is 66%.     

Cleavage of T4 species I ribonucleic acid by Escherichia coli ribonuclease III.

T4 Species I RNA, a molecule 140 nucleotides in length with some structural features very much like a tRNA, is specifically cleaved by an enzymatic activity in Escherichia coli extracts to give three segments with 19, 48 and 73 nucleotides. We report the purification and characterization of the E. coli RNase which cleaves two 3' phosphodiester bonds of T4 Species I RNA. This reaction has many properties in common with those catalyzed by E. coli RNase III, although the optimal salt conditions for T4 Species I RNA cleavage differ significantly from those for other RNase III-catalyzed reactions. The reaction is not catalyzed by extracts from an E. coli strain lacking RNase III activity. Furthermore, T4 Species I RNA is cleaved by highly purified E. coli RNase III to yield the same three specific fragments. We conclude that this specific cleavage is due to the action of RNase III, and that the requirement for lower ionic strength may reveal further important properties about this RNA processing enzyme.     

Swapping the domains of exoribonucleases RNase II and RNase R: conferring upon RNase II the ability to degrade ds RNA

RNase II and RNase R are the two E. coli exoribonucleases that belong to the RNase II super family of enzymes. They degrade RNA hydrolytically in the 3' to 5' direction in a processive and sequence independent manner. However, while RNase R is capable of degrading structured RNAs, the RNase II activity is impaired by dsRNAs. The final end-product of these two enzymes is also different, being 4 nt for RNase II and 2 nt for RNase R. RNase II and RNase R share structural properties, including 60% of amino acid sequence similarity and have a similar modular domain organization: two N-terminal cold shock domains (CSD1 and CSD2), one central RNB catalytic domain, and one C-terminal S1 domain. We have constructed hybrid proteins by swapping the domains between RNase II and RNase R to determine which are the responsible for the differences observed between RNase R and RNase II. The results obtained show that the S1 and RNB domains from RNase R in an RNase II context allow the degradation of double-stranded substrates and the appearance of the 2 nt long end-product. Moreover, the degradation of structured RNAs becomes tail-independent when the RNB domain from RNase R is no longer associated with the RNA binding domains (CSD and S1) of the genuine protein. Finally, we show that the RNase R C-terminal Lysine-rich region is involved in the degradation of double-stranded substrates in an RNase II context, probably by unwinding the substrate before it enters into the catalytic cavity.