Identification and characterization of the Escherichia coli rbn gene encoding the tRNA processing enzyme RNase BN.

The gene encoding RNase BN was localized to 88 min on the Escherichia coli chromosome by a novel suppressor assay and conjugational and transductional analysis. Assay of subclones derived from lambda phage 543 of the Kohara library, which encompasses this region of the chromosome, for elevated RNase BN activity identified o290, a previously reported open reading frame, as the gene encoding RNase BN. Interruption of this gene with a Kan(r) cassette and introduction into the chromosome eliminated cellular RNase BN activity but had no effect on cell growth. On the basis of these data, we suggest that o290 be renamed rbn. Potential homologs of rbn in other organisms also were identified.     

Purification and characterization of Escherichia coli RNase T

RNase T, a nuclease thought to be involved in end-turnover of tRNA, has been purified about 4,000-fold from extracts of Escherichia coli. At this stage of purification, the enzyme was judged to be at least 95% pure based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native molecular weight of RNase T determined from gel filtration and sedimentation analyses is about 50,000, whereas the monomer molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 25,000, suggesting that the protein is an alpha 2 dimer. Purified RNase T is extremely sensitive to inactivation by oxidation, sulfhydryl group reagents, and temperature. The ribonuclease activity against tRNA-C-C-[14C]A is optimal at pH 8-9 in the presence of 2-5 mM MgCl2 and ionic strengths of less than 50mM. Although RNase T is highly specific for intact tRNA-C-C-A as a substrate and can hydrolyze all species in a mixed population of tRNA, it is inhibited by other RNAs, such as poly(A), rRNA, 5 S RNA, and tRNA-C-C. RNase T is an exoribonuclease which initiates attack at a free 3' terminus of tRNA and releases AMP; aminoacyl-tRNA is not a substrate. The role of RNase T in the end-turnover of tRNA and its possible involvement in other aspects of RNA metabolism are discussed.     

Purification and characterization of the Escherichia coli exoribonuclease RNase R. Comparison with RNase II

Escherichia coli RNase R, a 3' --> 5' exoribonuclease homologous to RNase II, was overexpressed and purified to near homogeneity in its native untagged form by a rapid procedure. The purified enzyme was free of nucleic acid. It migrated upon gel filtration chromatography as a monomer with an apparent molecular mass of approximately 95 kDa, in close agreement with its expected size based on the sequence of the rnr gene. RNase R was most active at pH 7.5-9.5 in the presence of 0.1-0.5 mm Mg(2+) and 50-500 mm KCl. The enzyme shares many catalytic properties with RNase II. Both enzymes are nonspecific processive ribonucleases that release 5'-nucleotide monophosphates and leave a short undigested oligonucleotide core. However, whereas RNase R shortens RNA processively to di- and trinucleotides, RNase II becomes more distributive when the length of the substrate reaches approximately 10 nucleotides, and it leaves an undigested core of 3-5 nucleotides. Both enzymes work on substrates with a 3'-phosphate group. RNase R and RNase II are most active on synthetic homopolymers such as poly(A), but their substrate specificities differ. RNase II is more active on poly(A), whereas RNase R is much more active on rRNAs. Neither RNase R nor RNase II can degrade a complete RNA-RNA or DNA-RNA hybrid or one with a 4-nucleotide 3'-RNA overhang. RNase R differs from RNase II in that it cannot digest DNA oligomers and is not inhibited by such molecules, suggesting that it does not bind DNA. Although the in vivo function of RNase R is not known, its ability to digest certain natural RNAs may explain why it is maintained in E. coli together with RNase II.     

Purification and characterization of RNase P from Clostridium sporogenes

RNase P is a multi-subunit enzyme responsible for the accurate processing of the 5' terminus of all tRNAs. The RNA subunit from Clostridium sporogenes has been partially purified and characterized. The RNA is approximately 400 nucleotides long and makes a precise endonucleolytic cleavage at the mature 5' terminus of tRNA. The RNA requires moderate concentrations of Mg2+ (20 mM) and relatively high concentrations of NH4Cl (800 mM) for optimal activity. Mn2+ effectively substitutes for Mg2+ at 2 mM. Zn2+, Ni2+, Ca2+, and Co2+ are ineffective at stimulating activity. Monovalent ions are, in general, more effective the greater the ionic radius (NH+4 greater than Cs greater than Rb greater than K greater than Na). In contrast to the activity of Bacillus subtilis, C. sporogenes RNase P RNA is significant more active in (NH4)2SO4 than in NH4Cl.     

Catalytic Properties of RNase BN/RNase Z from Escherichia coli: RNase BN IS BOTH AN EXO- AND ENDORIBONUCLEASE*

Processing of the 3′ terminus of tRNA in many organisms is carried out by an endoribonuclease termed RNase Z or 3′-tRNase, which cleaves after the discriminator nucleotide to allow addition of the universal -CCA sequence. In some eubacteria, such as Escherichia coli, the -CCA sequence is encoded in all known tRNA genes. Nevertheless, an RNase Z homologue (RNase BN) is still present, even though its action is not needed for tRNA maturation. To help identify which RNA molecules might be potential substrates for RNase BN, we carried out a detailed examination of its specificity and catalytic potential using a variety of synthetic substrates. We show here that RNase BN is active on both double- and single-stranded RNA but that duplex RNA is preferred. The enzyme displays a profound base specificity, showing no activity on runs of C residues. RNase BN is strongly inhibited by the presence of a 3′-CCA sequence or a 3′-phosphoryl group. Digestion by RNase BN leads to 3-mers as the limit products, but the rate slows on molecules shorter than 10 nucleotides in length. Most interestingly, RNase BN acts as a distributive exoribonuclease on some substrates, releasing mononucleotides and a ladder of digestion products. However, RNase BN also cleaves endonucleolytically, releasing 3′ fragments as short as 4 nucleotides. Although the presence of a 3′-phosphoryl group abolishes exoribonuclease action, it has no effect on the endoribonucleolytic cleavages. These data suggest that RNase BN may differ from other members of the RNase Z family, and they provide important information to be considered in identifying a physiological role for this enzyme.Maturation of tRNA precursors requires the removal of 5′ and 3′ precursor-specific sequences to generate the mature, functional tRNA (1). In eukaryotes, archaea, and certain eubacteria, the 3′-processing step is carried out by an endoribonuclease termed RNase Z or 3′-tRNase (2–6). However, in some bacteria, such as Escherichia coli, removal of 3′ extra residues is catalyzed by any of a number of exoribonucleases (7, 8). The major determinant for which mode of 3′-processing is utilized appears to be whether or not the universal 3′-terminal CCA sequence is encoded (2, 9). Thus, for those tRNA precursors in which the CCA sequence is absent, endonucleolytic cleavage by RNase Z right after the discriminator nucleotide generates a substrate for subsequent CCA addition by tRNA nucleotidyltransferase (1–3, 10). In view of this role for RNase Z in 3′-tRNA maturation, it is surprising that E. coli, an organism in which the CCA sequence is encoded in all tRNA genes (2), nevertheless contains an RNase Z homologue (11), because its action would appear not to be necessary. In fact, the physiological function of this enzyme in E. coli remains unclear, because mutants lacking this protein have no obvious growth phenotype (12). Hence, there is considerable interest in understanding the enzymatic capabilities of this enzyme.The E. coli RNase Z homologue initially was identified as a zinc phosphodiesterase (11) encoded by the elaC gene (now called rbn) (13). Subsequent work showed that the protein also displayed endoribonuclease activity on certain tRNA precursors in vitro (6, 14). However, more recent studies revealed that this protein actually is RNase BN, an enzyme originally discovered in 1983 and shown to be essential for maturation of those bacteriophage T4 tRNA precursors that lack a CCA sequence (15, 16). Using synthetic mimics of these T4 tRNA precursors, RNase BN was found to remove their 3′-terminal residue as a mononucleotide to generate a substrate for tRNA nucleotidyltransferase. Based on these reactions RNase BN was originally thought to be an exoribonuclease (13, 15, 17). However, subsequent work by us and others showed that it can act as an endoribonuclease on tRNA precursors (13, 18). RNase BN is required for maturation of tRNA precursors in E. coli mutant strains devoid of all other 3′-tRNA maturation exoribonucleases, although it is the least efficient RNase in this regard (7, 19). Thus, under normal circumstances, it is unlikely that RNase BN functions in maturation of tRNA in vivo except in phage T4-infected cells (15, 16).To obtain additional information on what types of RNA molecules might be substrates for RNase BN and to clarify whether it is an exo- or endoribonuclease, we have carried out a detailed examination of its catalytic properties and substrate specificity. We show here that RNase BN has both exo- and endoribonuclease activity and that it can act on a wide variety of RNA substrates. These findings suggest that E. coli RNase BN may differ from other members of the RNase Z family of enzymes.     

A multiple mutant of Escherichia coli lacking the exoribonucleases RNase II, RNase D, and RNase BN

A multiple mutant strain of Escherichia coli containing mutations affecting the exoribonucleases, RNase II, RNase D, and RNase BN, and also the endonuclease, RNase I, was constructed by P1-mediated transduction. Extracts of the mutant strain were lacking the aforementioned RNase activities. The multiple mutant displayed normal growth in both rich and minimal media at a variety of temperatures, recovered from starvation essentially as the wild-type parent, and could support the growth of a variety of bacteriophages. In addition, RNA synthesis was normal and no precursor RNA accumulation was observed. The properties of the mutant strain indicate that the three exoribonucleases are not essential for the viability of E. coli. The implications of these findings to our understanding of RNA processing and degradation are discussed.     

Purification and characterization of a ketimine-reducing enzyme

An NAD(P)H-dependent reductase able to reduce a new class of cyclic unsaturated compounds named ketimines has been detected and purified 2500-fold from pig kidney. Some molecular and kinetic properties of this enzyme have been determined. The enzymatic reduction proceeds with a classical ping-pong mechanism and some results suggest that the true substrate has the ketiminic structure and is in equilibrium with the enaminic and keto-open forms. As previously described, ketimines arise from the deamination of a number of sulfur-containing amino acids, i.e. L-cystathionine, L-lanthionine and S-aminoethyl-L-cysteine, catalyzed by a widespread mammalian transaminase. The enzymatic reduction products of ketimines have been identified as cyclothionine, 1,4-thiomorpholine 3,5-dicarboxylic acid and 1,4-thiomorpholine 3-carboxylic acid. Some of these compounds have been detected in mammals, thus suggesting a possible role of this enzyme in their biosynthesis.     

Mode of Action of RNase BN/RNase Z on tRNA Precursors: RNase BN DOES NOT REMOVE THE CCA SEQUENCE FROM tRNA*

RNase BN, the Escherichia coli homolog of RNase Z, was previously shown to act as both a distributive exoribonuclease and an endoribonuclease on model RNA substrates and to be inhibited by the presence of a 3′-terminal CCA sequence. Here, we examined the mode of action of RNase BN on bacteriophage and bacterial tRNA precursors, particularly in light of a recent report suggesting that RNase BN removes CCA sequences (Takaku, H., and Nashimoto, M. (2008) Genes Cells 13, 1087–1097). We show that purified RNase BN can process both CCA-less and CCA-containing tRNA precursors. On CCA-less precursors, RNase BN cleaved endonucleolytically after the discriminator nucleotide to allow subsequent CCA addition. On CCA-containing precursors, RNase BN acted as either an exoribonuclease or endoribonuclease depending on the nature of the added divalent cation. Addition of Co²⁺ resulted in higher activity and predominantly exoribonucleolytic activity, whereas in the presence of Mg²⁺, RNase BN was primarily an endoribonuclease. In no case was any evidence obtained for removal of the CCA sequence. Certain tRNA precursors were extremely poor substrates under any conditions tested. These findings provide important information on the ability of RNase BN to process tRNA precursors and help explain the known physiological properties of this enzyme. In addition, they call into question the removal of CCA sequences by RNase BN.     

Exoribonuclease and Endoribonuclease Activities of RNase BN/RNase Z both Function in Vivo

Escherichia coli RNase BN, a member of the RNase Z family of endoribonucleases, differs from other family members in that it also can act as an exoribonuclease in vitro. Here, we examine whether this activity of RNase BN also functions in vivo. Comparison of the x-ray structure of RNase BN with that of Bacillus subtilis RNase Z, which lacks exoribonuclease activity, revealed that RNase BN has a narrower and more rigid channel downstream of the catalytic site. We hypothesized that this difference in the putative RNA exit channel might be responsible for the acquisition of exoribonuclease activity by RNase BN. Accordingly, we generated several mutant RNase BN proteins in which residues within a loop in this channel were converted to the corresponding residues present in B. subtilis RNase Z, thus widening the channel and increasing its flexibility. The resulting mutant RNase BN proteins had reduced or were essentially devoid of exoribonuclease activity in vitro. Substitution of one mutant rbn gene (P142G) for wild type rbn in the E. coli chromosome revealed that the exoribonuclease activity of RNase BN is not required for maturation of phage T4 tRNA precursors, a known specific function of this RNase. On the other hand, removal of the exoribonuclease activity of RNase BN in a cell lacking other processing RNases leads to slower growth and affects maturation of multiple tRNA precursors. These findings help explain how RNase BN can act as both an exo- and an endoribonuclease and also demonstrate that its exoribonuclease activity is capable of functioning in vivo, thus widening the potential role of this enzyme in E. coli.     

Purification and characterization of the D-alanyl-D-alanine-adding enzyme from Escherichia coli

The Escherichia coli D-alanyl-D-alanine-adding enzyme, which catalyzes the final cytoplasmic step in the biosynthesis of the bacterial peptidoglycan precursor UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-diaminopimelyl-D-Ala-D- Ala, has been purified to homogeneity from an E. coli strain that harbors a recombinant plasmid bearing the structural gene for this enzyme, murF. The enzyme is a monomer of molecular weight 49,000, and it has a turnover number of 784 min-1 for ATP-driven amide bond formation. Experiments monitoring the fate of radiolabeled UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-2,6-diaminopimelate and D-trifluoroalanine proved that the preceding enzyme in the D-alanine branch pathway, D-alanine:D-alanine ligase (ADP), is capable of synthesizing fluorinated dipeptides, which the D-Ala-D-Ala-adding enzyme can then incorporate to form UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-2,6-diaminopimelyl-D-++ +trifluoroAla-D- trifluoroAla.     

Purification and characterization of the sea urchin embryo hatching enzyme

The sea urchin hatching enzyme provides an interesting model for the control of gene expression during early development. In order to study its properties and developmental regulation, the hatching enzyme of the species Paracentrotus lividus has been purified. The fertilization envelopes of the embryos were digested before hatching by a crude culture supernatant previously made. The enzyme was then solubilized by 1 M NaCl and 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and purified by hydrophobic chromatography on Procion-agarose. A 470-fold increase in specific activity was obtained. The kinetic parameters of the proteolytic activity using dimethylcasein as substrate are: Km = 120 micrograms x ml-1, Vm = 200 mumol x min-1 x mg-1, and kcat = 180 s-1 at 500 mM NaCl, 10 mM CaCl2, pH 8.0, at 35 degrees C. The purified enzyme is highly active on fertilization envelopes: at 20 degrees C and 500 mM NaCl, 10 mM CaCl2, pH 8.0, 100 ng of enzyme completely denudes embryos in about 20 min under standard conditions. The molecular mass of the enzyme was estimated as 57 kDa by gel filtration, 51 kDa by gel electrophoresis, and 52 kDa by amino acid analysis. The hatching enzyme was shown to be a glycoprotein which autolyzes to a 30-kDa inactive form. Antibodies raised against the 51- or 30-kDa forms reacted with both these forms. Immunoblotting experiments showed that the hatching supernatants contain important amounts of the autolyzed species.     

Purification and characterization of the elastase-like enzyme of the bovine granulocyte

The extracts of granules isolated from bovine granulocytes show elastase- and chymotrypsin-like activities, as detected with specific synthetic substrates. Extraction of these enzymes depends upon salt concentration. In the course of the present studies a 21-fold purification of the elastase-like enzyme was achieved on a (Ala)3-CH-Sepharose 4B gel. The molecular weight of the enzyme is 33 000, as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. The elastase-like activity is inhibited by phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, basic pancreatic inhibitor and by heparin at different rates. Elastatinal inhibits the enzyme competitively (Ki = 80 microM). The cytosol of bovine granulocytes contains a protein which strongly inhibits the elastase-like enzyme of the bovine granulocyte (Ki = 0.4 nM) as well as porcine pancreatic elastase (Ki = 11 nM).     

The RNase Z homologue encoded by Escherichia coli elaC gene is RNase BN

In eukaryotes, archaea, and in some eubacteria, removal of 3' precursor sequences during maturation of tRNA is catalyzed by an endoribonuclease, termed RNase Z. In contrast, in Escherichia coli, a variety of exoribonucleases carry out final 3' maturation. Yet, E. coli retains an RNase Z homologue, ElaC, whose function is under active study. We have overexpressed and purified to homogeneity His-tagged ElaC and show here that it is, in fact, the previously described enzyme, RNase BN. Thus, purified ElaC displays structural and catalytic properties identical to those ascribed to RNase BN. In addition, an elaC mutant strain behaves identically to a known RNase BN- strain, CAN. Finally, we show that wild type elaC can complement the mutation in strain CAN and that the elaC gene in strain CAN carries a nonsense mutation that results in loss of RNase BN activity. These data correct a previous misassignment for the gene encoding RNase BN. Based on the fact that the original RNase BN mutation has now been identified, we propose that the elaC gene be renamed rbn.     

Purification, molecular and enzymic characterization of an acid RNase from the insect Ceratitis capitata

An acid ribonuclease has been purified from the insect Ceratitis capitata. The specific activity of the purified enzyme is 580 units/mg. This enzyme is a single polypeptide chain of about 35.5 kDa, containing only one disulfide bridge and no free -SH groups. The A0.1%1cm at 280 nm is 1.90. The hydrodynamic radius of the native enzyme is 2.5 nm. The secondary structure of this RNase is composed of 10% alpha-helix, 31% beta-structure and 59% aperiodic conformation with an average number of residues per helical segment of 10, based on circular dichroic measurements. Optimum parameters for the enzyme activity are pH 5.5, 0.15 M ionic strength and 40 degrees C. Divalent cations are not required for the enzymic catalysis. This enzyme has been characterized as cyclizing endoribonuclease.     

Brain arylamidase. Purification and characterization of the soluble bovine enzyme

1. An enzyme acting on aminoacyl-β-naphthylamides has been isolated from the soluble fraction of bovine brain and purified 205-fold by means of ammonium sulphate fractionation, hydroxyapatite adsorption and DEAE-Sephadex column chromatography. 2. Arylamidase requires thiol groups for retention of its activity, is heat-labile and is susceptible to freezing. p-Chloromercuribenzoate and N-ethylmaleimide inactivate the enzyme rapidly. 3. Metal ions are not required for its activity, but stimulation by Mn²⁺ and Mg²⁺ and inactivation by Co²⁺ and Zn²⁺ are observed. 4. Optimum pH7·5 in phosphate buffer was exhibited for all substrates tested except l-leucyl-β-naphthylamide, for which optimum pH is 6·5. 5. K_m values for a number of substrates have been obtained and substrate inhibition at high concentrations was demonstrated. 6. The molecular weight is approx. 70000 as determined by Sephadex-gel filtration.     

Escherichia coli RNase D. Purification and structural characterization of a putative processing nuclease

RNase D, a putative tRNa processing nuclease, has been purified about 1,000-fold from extracts of Escherichia coli to apparent homogeneity, as judged by acrylamide gel electrophoresis under nondenaturing and denaturing conditions and by gel electrofocusing. The purified enzyme is a single chain protein with a molecular weight of 40,000 and an isoelectric point of about 6.2. Spectral analysis indicated that RNase D is devoid of nucleic acid. Amino acid analysis suggested a low content of cysteine, and this was confirmed by the relative insensitivity of the enzyme to sulfhydryl group reagents. RNase D is sensitive to inactivation by elevated temperatures but can be protected by a variety of RNAs, including those which are not substrates for hydrolysis. The relation of RNase D to other known E. coli ribonucleases and to other previously identified processing activities, is discussed.     

Purification and characterization of a staphylolytic enzyme from Pseudomonas aeruginosa

A strain of Pseudomonas aeruginosa has been shown to produce an enzyme that lyses viable cells of Staphylococcus aureus. The maximal yield of the enzyme was obtained from shake flask cultures of P. aeruginosa which were grown for 18 to 22 hr at 37 C in Trypticase Soy Broth. A 333-fold purification of the enzyme was obtained by acetone precipitation of the culture liquor, followed by column chromatography on phosphonic acid cellulose and Bio-Gel P2. The staphylolytic enzyme exhibited maximal activity at 37 C in 0.01 m sodium phosphate (pH 8.5) and was stable at 37 C in the pH range of 7.5 to 9.5. The inhibition and stabilization of the enzyme by various organic and inorganic materials was investigated. Spheroplasts of S. aureus were formed by treating viable cells with the staphylolytic enzyme in 1 m sucrose or human serum.