Angiogenin is a cytotoxic, tRNA-specific ribonuclease in the RNase A superfamily.

Angiogenin is a 14.4-kDa human plasma protein with 65% homology to RNase A that retains the key active site residues and three of the four RNase A disulfide bonds. We demonstrate that recombinant angiogenin functions as a cytotoxic tRNA-specific RNase in cell-free lysates and when injected into Xenopus oocytes. Inhibition of protein synthesis by angiogenin correlates with degradation of endogenous oocyte tRNA. Exogenous, radiolabeled tRNA is also hydrolyzed by angiogenin, whereas oocyte rRNA and mRNA are not detectably degraded by angiogenin. Protein synthesis was restored to angiogenin-injected oocytes by injecting the RNase inhibitor RNasin plus total Xenopus or calf liver tRNAs, thereby demonstrating that the tRNA degradation induced by angiogenin was the sole cause of cytotoxicity. A similar tRNA-reversible inhibition of protein synthesis was seen in rabbit reticulocyte lysates. Angiogenin therefore appears to be a specific cellular tRNase, whereas five homologues in the RNase A superfamily lack angiogenin's specificity for tRNA. One of these homologues purified from human eosinophils, eosinophil-derived neurotoxin, nonspecifically degrades oocyte RNA similar to RNase A and is also cytotoxic at very low concentrations.     

A covalent angiogenin/ribonuclease hybrid with a fourth disulfide bond generated by regional mutagenesis

Human angiogenin is a blood vessel inducing protein whose primary structure displays 33% identity to that of bovine pancreatic ribonuclease A (RNase A). Angiogenin catalyzes limited cleavage of 18S and 28S ribosomal RNA and is several orders of magnitude less potent than RNase A toward conventional substrates. A striking structural difference between angiogenin and RNase is the virtual absence of sequence similarity within the region of RNase that contains the Cys-65--Cys-72 disulfide bond. Indeed, angiogenin lacks this disulfide linkage. The present report describes the use of regional mutagenesis to generate a covalent angiogenin/RNase hybrid protein, ARH-I, where residues 58-70 of angiogenin have been replaced by the corresponding segment of RNase A (residues 59-73). The protein expressed in Escherichia coli readily folds at pH 8.5 to form the four expected disulfide bonds. The in vivo angiogenic potency of ARH-I is markedly diminished compared with that of angiogenin when examined using the chick chorioallantoic membrane assay. In contrast, its enzymatic activity is dramatically increased. With high molecular weight wheat germ RNA and tRNA, ARH-I is 660- and 300-fold more active than angiogenin, respectively, while with poly(uridylic acid), poly(cytidylic acid), cytidylyl(3'----5')adenosine (CpA), and uridylyl(3'----5')adenosine (UpA) activity is enhanced by about 200-fold. In addition, the specificity of ARH-I toward dinucleoside 3',5'-phosphates is qualitatively similar to RNase A; while angiogenin prefers cytidylyl(3'----5')guanosine (CpG) to UpA, both RNase and the hybrid prefer UpA to CpG. ARH-I also displays greater than 10-fold enhanced activity toward rRNA in intact ribosomes, while abolishing the capacity of the ribosome to support cell-free protein synthesis. The enhanced enzymatic properties of ARH-I parallel a 2-fold increase in chemical reactivity of active-site lysine and histidine residues based on rates of chemical modification. The data indicate that introduction of a region of RNase A containing the Cys-65--Cys-72 disulfide bond into angiogenin dramatically increases RNase-like enzymatic activity while reducing its angiogenicity.     

Replacement of residues 8-22 of angiogenin with 7-21 of RNase A selectively affects protein synthesis inhibition and angiogenesis.

The region of human angiogenin containing residues 8-21 is highly conserved in angiogenins from four mammalian species but differs substantially from the corresponding region of the homologous protein ribonuclease A (RNase A). Regional mutagenesis has been employed to replace this segment of angiogenin with the corresponding RNase A sequence, and the activities of the resulting covalent angiogenin/RNase hybrid, designated ARH-III, have been examined. The ribonucleolytic activity of ARH-III is unchanged toward most substrates, including tRNA, naked 18S and 28S rRNA, CpA, CpG, UpA, and UpG. In contrast, the capacity of ARH-III to inhibit cell-free protein synthesis is decreased 20-30-fold compared to that of angiogenin. The angiogenic activity of ARH-III is also different; it is actually more potent. It induces a maximal response in the chick chorioallantoic membrane assay at 0.1 ng per egg, a 10-fold lower dose than required for angiogenin. In addition, binding of ARH-III to the placental ribonuclease inhibitor is increased by at least 1 order of magnitude (Ki less than or equal to 7 x 10(-17) M) compared to angiogenin. Thus, mutation of a highly conserved region of angiogenin markedly affects those properties likely involved in its biological function(s); it does not, however, alter ribonucleolytic activity toward most substrates.     

Kinetic characterization of two active mutants of placental ribonuclease inhibitor that lack internal repeats

Human placental ribonuclease inhibitor (PRI), a 50-kDa tight-binding inhibitor of angiogenin and pancreatic ribonuclease, consists predominantly of 7 internal repeats, each 57 residues long. Repeats 3 plus 4 (residues 144-257) or repeat 6 (residues 315-371) can be deleted to give mutant proteins, PRI delta 3-4 and PRI delta 6, respectively, that retain inhibitory activity [Lee, F. S., & Vallee, B. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1879-1883]. We describe here the isolation and characterization of these two active mutant proteins. Both inhibit the enzymatic activities of either angiogenin or bovine pancreatic ribonuclease A (RNase A) with a 1:1 stoichiometry, and the mode of inhibition of RNase A by either is competitive. PRI delta 3-4 binds to angiogenin and RNase A with Ki values of 0.72 and 170 pM, respectively The corresponding values for PRI delta 6 are 22 and 43 pM, respectively. Since recombinant PRI to angiogenin and RNase A with Ki values of 0.29 and 68 fM, respectively, deletion of repeats 3 plus 4 weakens both interactions 2500-fold while deletion of repeat 6 weakens them 76,000- and 630-fold, respectively. Therefore, either the deletion of these repeats has altered the conformation of the angiogenin/RNase binding site in PRI or the deleted repeats contribute directly to the binding site, or both. In addition, the tighter binding to angiogenin versus RNase A seen with native PRI has been preserved in PRI delta 3-4 but has been almost completely abolished in PRI delta 6.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stress induces tRNA cleavage by angiogenin in mammalian cells

tRNAs play a central role in protein translation, acting as the carrier of amino acids. By cloning microRNAs, we unexpectedly obtained some tRNA fragments generated by tRNA cleavage in the anticodon loop. These tRNA fragments are present in many cell lines and different mouse tissues. In addition, various stress conditions can induce this tRNA cleavage event in mammalian cells. More importantly, angiogenin (ANG), a member of RNase A superfamily, appears to be the nuclease which cleaves tRNAs into tRNA halves in vitro and in vivo. These results imply that angiogenin plays an important physiological role in cell stress response, except for the known function of inducing angiogenesis.     

Tight-binding inhibition of angiogenin and ribonuclease A by placental ribonuclease inhibitor

The dissociation rate constant of the angiogenin-placental ribonuclease inhibitor complex was determined by measuring the release of free angiogenin from the complex in the presence of scavenger for free placental ribonuclease inhibitor (PRI). In 0.1 M NaCl, pH 6, 25 degrees C, this value is 1.3 X 10(-7) s-1 (t1/2 congruent to 60 days). The Ki value for the binding of PRI to angiogenin, calculated from the association and dissociation rate constants, is 7.1 X 10(-16) M. The corresponding values for the interaction of RNase A with PRI, determined by similar means, are both considerably higher: the dissociation rate constant is 1.5 X 10(-5) s-1 (t1/2 = 13 h), and the Ki value is 4.4 X 10(-14) M. Thus, PRI binds about 60 times more tightly to angiogenin than to RNase A. The effect of increasing sodium chloride concentration on the binding of PRI to RNase A was explored by Henderson plots. The Ki value increases to 39 pM in 0.5 M NaCl and to 950 pM in 1 M NaCl, suggesting the importance of ionic interactions. The mode of inhibition of RNase A by PRI was determined by examining the effect of a competitive inhibitor of RNase A, cytidine 2'-phosphate, on the association rate of PRI with RNase A. Increasing concentrations of cytidine 2'-phosphate decrease the association rate in a manner consistent with a competitive mode of inhibition.     

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.     

Design and isolation of ribozyme-substrate pairs using RNase P-based ribozymes containing altered substrate binding sites.

Substrate recognition and cleavage by the bacterial RNase P RNA requires two domains, a specificity domain, or S-domain, and a catalytic domain, or C-domain. The S-domain binds the T stem-loop region in a pre-tRNA substrate to confer specificity for tRNA substrates. In this work, the entire S-domain of the Bacillus subtilis RNase P RNA is replaced with an artificial substrate binding module. New RNA substrates are isolated by in vitro selection using two libraries containing random regions of 60 nt. At the end of the selection, the cleavage rates of the substrate library are approximately 0.7 min(-1)in 10 mM MgCl(2)at 37 degrees C, approximately 4-fold better than the cleavage of a pre-tRNA substrate by the wild-type RNase P RNA under the same conditions. The contribution of the S-domain replacement to the catalytic efficiency is from 6- to 22 000-fold. Chemical and nuclease mapping of two ribozyme-product complexes shows that this contribution correlates with direct interactions between the S-domain replacement and the selected substrate. These results demonstrate the feasibility of design and isolation of RNase P-based, matching ribozyme-substrate pairs without prior knowledge of the sequence or structure of the interactive modules in the ribozyme or substrate.     

Isolation and characterization of angiogenin-1 and a novel protein designated lactogenin from bovine milk.

This paper reports the isolation and characterization from bovine milk of two proteins: angiogenin-1, a recently discovered angiogenin, and lactogenin, a novel protein. Both proteins were adsorbed on and eluted closely from CM-Sepharose and Mono S. Lactogenin possessed a molecular weight (17 kDa) slightly higher than that of angiogenin-1 (15 kDa). Lactogenin had a higher ribonucleolytic (RNase) activity than angiogenin-1 towards yeast transfer RNA (tRNA). The Km values estimated for the RNase activities of angiogenin-1 and lactogenin were 51 microM and 40 microM respectively. Both were specific for poly C. The optimal pH for the RNase activities of angiogenin-1 and lactogenin was 7.75 and 7.5 respectively. Comparison of the amino acid sequences of cyanogen bromide fragments and the pyroglutaminase-treated N-terminal fragment of lactogenin with the sequence of bovine liver RNase (RNase BL4) revealed identity in residues 3-22, 24, 26-27, 37, 41-44, 46-50, 54, 56, 63, 72-80, and 83. Considerable similarity to the N-terminal sequence of angiogenin-2 was also noted. Both lactogenin and angiogenin-1 inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC(50) below 100 nM.     

Tryptophan fluorescence as a probe of placental ribonuclease inhibitor binding to angiogenin

The binding of human placental ribonuclease inhibitor (PRI) to angiogenin, a human protein that induces neovascularization, occurs with a 1:1 stoichiometry and is accompanied by a 50% increase in tryptophan fluorescence. In contrast, the binding of PRI to bovine pancreatic RNase A or to angiogenin oxidized at its single tryptophan residue results in a quenching of fluorescence. These observations suggest that there is a change in the local environment of Trp-89 of angiogenin. Quenching experiments with acrylamide are consistent with the view that Trp-89 is exposed in the native protein and becomes less accessible upon formation of the complex with PRI. Stopped-flow kinetic measurements monitoring the fluorescence enhancement indicate a two-step mechanism for the binding of PRI to angiogenin. The first step involves rapid formation of an enzyme-inhibitor complex, EI, followed by a slower isomerization of EI to a tight enzyme-inhibitor complex, EI*: (Formula: see text). In 0.1 M NaCl at pH 6 and 25 degrees C, the values of K1 and K2 are 0.53 microM and 97 s-1, respectively. The apparent second-order rate constant of association at protein concentrations much less than K1 is approximated by K2/K1 and equals 1.8 X 10(8) M-1 s-1. The corresponding value for the association of PRI with RNase A is only slightly higher, 3.4 X 10(8) M-1 s-1. The effects of pH and sodium chloride concentration on the association rate of PRI with angiogenin suggest the importance of ionizable groups and ionic interactions, respectively, in the association process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of human placental ribonuclease inhibitor in Escherichia coli

Human placental ribonuclease inhibitor (PRI) has been expressed in and isolated from Escherichia coli. Its apparent molecular weight, immunoreactivity and amino acid composition are virtually identical with those of native PRI. It inhibits the enzymatic activities of either angiogenin, a blood vessel inducing protein homologous to pancreatic RNase (RNase A), or RNase A in a stoichiometry of 1:1. Recombinant PRI binds to angiogenin and RNase A with Ki values of 2.9 x 10(-16) M and 6.8 x 10(-14) M, respectively, comparable to the affinities of native PRI for these enzymes. Thus, these results confirm that PRI inhibits angiogenin more effectively than RNase A.     

Functional expression of recombinant human ribonuclease/angiogenin inhibitor in stably transformed <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> S2 cells

A recombinant plasmid harboring heterologous genes coding human ribonuclease/angiogenin inhibitor (RAI) was expressed in stably transformed Drosophila melanogaster Schneider 2 (S2) cells. Stably transformed polyclonal cell populations expressing RAI were isolated after 4 weeks of selection with hygromycin B. Recombinant RAI with a molecular weight of 50 kDa was detected in the intracellular (cell) and extracellular (medium) fractions of S2 cells. Recombinant RAI was purified from the extracellular fraction using a two-step purification scheme comprised of Ni-NTA and ion-exchange chromatography. Purified RAI migrated on SDS-PAGE as a single band in the elution fraction containing 300 mM NaCl. The ribonuclease inhibitor activity of purified RAI was measured using yeast tRNA and RNase A. Purified RAI exhibited an activity of ∼8 U μg⁻¹ for the inhibition of RNA degradation by RNase A. Cultivation of stably transformed S2 cells using HyQ^®SFX-insect MP medium increased cell growth by 79% and approximately doubled the production of recombinant RAI.     

A hybrid of bovine pancreatic ribonuclease and human angiogenin: an external loop as a module controlling substrate specificity?

A comparison of the sequences of three homologous ribonucleases (RNase A, angiogenin and bovine seminal RNase) identifies three surface loops that are highly variable between the three proteins. Two hypotheses were contrasted: (i) that this variation might be responsible for the different catalytic activities of the three proteins; and (ii) that this variation is simply an example of surface loops undergoing rapid neutral divergence in sequence. Three hybrids of angiogenin and bovine pancreatic ribonuclease (RNase) A were prepared where regions in these loops taken from angiogenin were inserted into RNase A. Two of the three hybrids had unremarkable catalytic properties. However, the RNase A mutant containing residues 63-74 of angiogenin had greatly diminished catalytic activity against uridylyl-(3'----5')-adenosine (UpA), and slightly increased catalytic activity as an inhibitor of translation in vitro. Both catalytic behaviors are characteristic of angiogenin. This is one of the first examples of an engineered external loop in a protein. Further, these results are complementary to those recently obtained from the complementary experiment, where residues 59-70 of RNase were inserted into angiogenin [Harper and Vallee (1989) Biochemistry, 28, 1875-1884]. Thus, the external loop in residues 63-74 of RNase A appears to behave, at least in part, as an interchangeable 'module' that influences substrate specificity in an enzyme in a way that is isolated from the influences of other regions in the protein.     

3'-N-Alkylamino-3'-deoxy-ara-uridines: a new class of potential inhibitors of ribonuclease A and angiogenin

In this study, we report the inhibition of ribonuclease A (RNase A) by certain aminonucleosides. This is the first such instance of the use of this group of compounds to investigate the inhibitory activity of this protein. The compounds synthesized have been tested for their ability to inhibit the ribonucleolytic activity of RNase A by an agarose gel-based assay. A tRNA precipitation assay and inhibition kinetic studies with cytidine 2',3'-cyclic monophosphate as the substrate have also been conducted for two of the compounds. Results indicate substantial inhibitory activity with inhibition association constants in the micromolar range. The experimental studies have been substantiated by docking of the aminonucleoside ligands to RNase A using AutoDock. We find that the ligands preferentially bind to the active site of the protein molecule with a favorable free energy of binding. The study has been extended to a member of the ribonuclease superfamily, angiogenin, which is a potent inducer of blood vessel formation. We show that the aminonucleosides act as potent inhibitors of angiogenin induced angiogenesis.     

Angiogenin cleaves tRNA and promotes stress-induced translational repression

Stress-induced phosphorylation of eIF2α inhibits global protein synthesis to conserve energy for repair of stress-induced damage. Stress-induced translational arrest is observed in cells expressing a nonphosphorylatable eIF2α mutant (S51A), which indicates the existence of an alternative pathway of translational control. In this paper, we show that arsenite, heat shock, or ultraviolet irradiation promotes transfer RNA (tRNA) cleavage and accumulation of tRNA-derived, stress-induced small RNAs (tiRNAs). We show that angiogenin, a secreted ribonuclease, is required for stress-induced production of tiRNAs. Knockdown of angiogenin, but not related ribonucleases, inhibits arsenite-induced tiRNA production and translational arrest. In contrast, knockdown of the angiogenin inhibitor RNH1 enhances tiRNA production and promotes arsenite-induced translational arrest. Moreover, recombinant angiogenin, but not RNase 4 or RNase A, induces tiRNA production and inhibits protein synthesis in the absence of exogenous stress. Finally, transfection of angiogenin-induced tiRNAs promotes phospho-eIF2α–independent translational arrest. Our results introduce angiogenin and tiRNAs as components of a phospho-eIF2α–independent stress response program.     

Enzymatically active angiogenin/ribonuclease A hybrids formed by peptide interchange

The primary structures of the blood vessel inducing protein human angiogenin and human pancreatic ribonuclease (RNase) are 35% identical. Angiogenin catalyzes the limited cleavage of ribosomal RNA (18 and 28 S), yielding a characteristic pattern of polynucleotide products, but shows no significant activity toward conventional pancreatic RNase substrates [Shapiro, R., Riordan, J. F., & Vallee, B. L. (1986) Biochemistry 25, 3527-3532]. Angiogenin/RNase hybrid enzymes--wherein particular regions of primary structure in RNase are replaced by the corresponding segments of angiogenin--serve to explore the structural features underlying angiogenin's characteristic activities. Herein we show that synthetic angiogenin peptides, Ang(1-21) and Ang(108-123), form noncovalent complexes with inactive fragments of bovine RNase A--RNase(21-124) (i.e., S-protein) and RNase(1-118), respectively--with regeneration of activity toward conventional RNase substrates. Maximal activities for the Ang(1-21)/S-protein complex (Kd = 1.0 microM) are 52%, 45%, and 15% toward cytidine cyclic 2',3'-phosphate, cytidylyl(3'----5')adenosine, and yeast RNA, respectively. In contrast, activities of the RNase(1-118)/Ang(108-123) hybrid (Kd = 25 microM) are 1-2 orders of magnitude lower toward cyclic nucleotides and dinucleoside phosphates. However, substitution of phenylalanine for Leu-115 in Ang(108-123) increases activity up to 100-fold. Both His-13 and His-114 in the angiogenin peptides are required for activity since their substitution by alanine yields inactive complexes. Importantly, the pattern of polynucleotide products formed during cleavage of ribosomal RNA by the Ang(1-21)/S-protein hybrid shows a striking resemblance to that formed by angiogenin, demonstrating that the hybrid retains features of both angiogenin and RNase A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partial purification and characterization of the components of the neutral ribonuclease II-inhibitor system of normal and distrophic mouse skeletal muscle

The complexes between a proteinaceous inhibitor and neutral ribonuclease II (EC 3.127.5) purified from low ionic strength extracts of normal and dystrophic mouse muscle are essentially indistinguishable in (a) purification behavior, (b) apparent molecular weights of approximately 50 000, (c) thermal denaturation (50% loss of activity in 5 min at 73.5 degrees C), (d) isoelectric points (pH 4.8), and (e) procedures for reversible resolution into free inhibitor and free RNase II. The free RNase II species are also similar whether obtained by resolution of the purified complexes or by direct isolation of free enzyme from dystrophic muscle. All have apparent molecular weights of 11 500 compared with 13 700 for bovine pancreatic RNase A; all retain 80% of activity after 5 min at 95 degrees C. The active RNase II prepared directly from muscle, by resolution of inhibitor complexes or by organic mercurial treatment of the inhibitor complexes, all have identical pH-activity profiles in 200 mM KC1 with an optimum near pH 7.0. In comparison RNase A has an optimum pH near 7.5 and its activity decreases more rapidly as KC1 concentration is increased above 50 mM KC1. RNase II inhibitor obtained by resolution of the purified complexes or by direct isolation in the free form from normal muscle extracts has an apparent molecular weight of 42 000 and is very sensitive to heat; it loses all activity at 40 degrees C in 5 min. These studies (a) provide methods for obtaining useful amounts of the components of the neutral RNase II - inhibitor system from muscle, (b) provide the first method reported for the reversible resolution of RNase II - inhibitor complexes, (c) fail to show any distinct difference between corresponding components of the system from normal and dystrophic mice, (d) establish interesting differences between the apparently homologous enzymes, murine muscle neutral RNase II, and bovine pancreatic RNase A, and (e) provide a substantially lower molecular weight estimate for RNase II inhibitor from muscle than has been reported for the inhibitor from liver, kidney, and placenta.     

Mutagenesis of residues flanking Lys-40 enhances the enzymatic activity and reduces the angiogenic potency of angiogenin.

The primary structure of the blood vessel inducing protein angiogenin is 35% identical with that of pancreatic ribonuclease (RNase) and contains counterparts for the critical RNase active-site residues His-12, Lys-41, and His-119. Although angiogenin is a ribonucleolytic enzyme, its activity toward conventional substrates is lower than that of pancreatic RNase by several orders of magnitude. Comparison of the amino acid sequences of RNase and angiogenin reveals several striking differences in the region flanking the active-site lysine, including a deletion and a transposition of aspartic acid and proline residues. In order to examine how these sequence changes alter the functional properties of angiogenin, an angiogenin/RNase hybrid protein (ARH-II), in which residues 38-41 of angiogenin (Pro-Cys-Lys-Asp) have been replaced by the corresponding segment of bovine pancreatic RNase (Asp-Arg-Cys-Lys-Pro), was prepared by regional mutagenesis. Compared to angiogenin, ARH-II has markedly diminished angiogenic activity on the chick embryo chorioallantoic membrane but 5-75-fold greater enzymatic activity toward a variety of polynucleotide and dinucleotide substrates. In addition, the specificity of ARH-II toward dinucleotide substrates differs from that of angiogenin and is qualitatively similar to that of pancreatic RNase. Thus, non-active-site residues near Lys-40 in angiogenin appear to play a significant role in determining enzymatic specificity and reactivity as well as angiogenic potency. An additional angiogenin/RNase hybrid protein (ARH-IV), in which residues 59-71 of ARH-II have been replaced by the corresponding segment of pancreatic RNase, was also prepared.(ABSTRACT TRUNCATED AT 250 WORDS)     

Product release is a rate-limiting step during cleavage by the catalytic RNA subunit of Escherichia coli RNase P.

The kinetic constants for cleavage of the tRNA(Tyr)Su3 precursor by the M1 RNA of E. coli RNase P were determined in the absence and presence of the C5 protein under single and multiple (steady state) turnover conditions. The rate constant of cleavage in the reaction catalyzed by M1 RNA alone was 5 times higher in single turnover than in multiple turnovers, suggesting that a rate-limiting step is product release. Cleavage by M1 RNA alone and by the holoenzyme under identical buffer conditions demonstrated that C5 facilitated product release. Addition of different product-like molecules under single turnover reaction conditions inhibited cleavage both in the absence and presence of C5. In the presence of C5, the Ki value for matured tRNA was approximately 20 times higher than in its absence, suggesting that C5 also reduces the interaction between the 5'-matured tRNA and the enzyme. In a growing cell the number of tRNA molecules is approximately 1000 times higher than the number of RNase P molecules. A 100-fold excess of matured tRNA over enzyme clearly inhibited cleavage in vitro. We discuss the possibility that RNase P is involved in the regulation of tRNA expression under certain growth conditions.     

Cleavage of 3',5'-pyrophosphate-linked dinucleotides by ribonuclease A and angiogenin

Recently, 3',5'-pyrophosphate-linked 2'-deoxyribodinucleotides were shown to be >100-fold more effective inhibitors of RNase A superfamily enzymes than were the corresponding monophosphate-linked (i.e., standard) dinucleotides. Here, we have investigated two ribo analogues of these compounds, cytidine 3'-pyrophosphate (P'-->5') adenosine (CppA) and uridine 3'-pyrophosphate (P'-->5') adenosine (UppA), as potential substrates for RNase A and angiogenin. CppA and UppA are cleaved efficiently by RNase A, yielding as products 5'-AMP and cytidine or uridine cyclic 2',3'-phosphate. The k(cat)/K(m) values are only 4-fold smaller than for the standard dinucleotides CpA and UpA, and the K(m) values (10-16 microM) are lower than those reported for any earlier small substrates (e.g., 500-700 microM for CpA and UpA). The k(cat)/K(m) value for CppA with angiogenin is also only severalfold smaller than for CpA, but the effect of lengthening the internucleotide linkage on K(m) is more modest. Ribonucleotide 3',5'-pyrophosphate linkages were proposed previously to exist in nature as chemically labile intermediates in the pathway for the generation of cyclic 2',3'-phosphate termini in various RNAs. We demonstrate that in fact they are relatively stable (t(1/2) > 15 days for uncatalyzed degradation of UppA at pH 6 and 25 degrees C) and that cleavage in vivo is most likely enzymatic. Replacements of the RNase A catalytic residues His12 and His119 by alanine reduce activity toward UppA by approximately 10(5)-and 10(3.3)-fold, respectively. Thus, both residues play important roles. His12 probably acts as a base catalyst in cleavage of UppA (as with RNA). However, the major function of His119 in RNA cleavage, protonation of the 5'-O leaving group, is not required for UppA cleavage because the pK(a) of the leaving group is much lower than that for RNA substrates. A crystal structure of the complex of RNase A with 2'-deoxyuridine 3'-pyrophosphate (P'-->5') adenosine (dUppA), determined at 1.7 A resolution, together with models of the UppA complex based on this structure suggest that His119 contributes to UppA cleavage through a hydrogen bond with a nonbridging oxygen atom in the pyrophosphate and through pi-pi stacking with the six-membered ring of adenine.