Solubilization and promoter analysis of RNA polymerase from rice stripe virus.

The RNA-dependent RNA polymerase associated with rice stripe virus was dissociated from viral RNA (vRNA) by CsCl centrifugation. The solubilized RNA-free RNA polymerase transcribed a model RNA template 50 nucleotides in length carrying the 5'- and 3'-terminal conserved sequences of all four genome RNA segments. A 3'-terminal half molecule of the model template was also active as a template. Hence, we propose that the 3'-terminal conserved sequence serves as a promoter for the rice stripe virus-associated RNA polymerase. The solubilized enzyme, however, was unable to transcribe vRNA. The failure of the solubilized enzyme to transcribe vRNA is discussed in relation to the apparent loss of RNA polymerase activity after treatment of virions with high concentrations of salt.     

Isolation and some properties of a deoxyribonuclease from Turbo cornutus.

1. Four DNases were found in the dried liver extract of a top shell, Turbo cornutus. The major one was purified 120-fold by phosphocellulose column chromatography, sulfoethylcellulose column chromatography and gel-filtration on Sephadex G-150. The yield was 2.7%. 2. The enzyme activity was not affected by Mg2+ (10(-3)--10(-2)M), EDTA (10(-3)--10(-2)M), or NaCl (10(-1)M). It showed a pH optimum of 4.7--4.8. Ionic strength was found to be critical for the maximal activity. The isoelectric point was 8.5--9.0. On heating at 50 degrees C C for 5 min the enzymic activity fell to half the initial value. 3. The enzyme preparation degraded native as well as heat-denatured DNA, but not RNA. It degraded heat-denatured DNA endonucleolytically to give oligonucleotides with 3'-phosphates. 4. The 3'-phosphate and 5'-hydroxy termini of oligonucleotides were investigated. At both the 3'- and 5'-terminal positions, purine nucleotides were predominant.     

Transcription strategy in a Closterovirus: a novel 5'-proximal controller element of Citrus Tristeza Virus produces 5'- and 3'-terminal subgenomic RNAs and differs from 3' open reading frame controller elements

Citrus tristeza virus (CTV) produces more than thirty 3'- or 5'-terminal subgenomic RNAs (sgRNAs) that accumulate to various extents during replication in protoplasts and plants. Among the most unusual species are two abundant populations of small 5'-terminal sgRNAs of approximately 800 nucleotides (nt) termed low-molecular-weight tristeza (LMT1 and LMT2) RNAs. Remarkably, CTV replicons with all 10 3' genes deleted produce only the larger LMT1 RNAs. These 5'-terminal positive-sense sgRNAs do not have corresponding negative strands and were hypothesized to be produced by premature termination during plus-strand genomic RNA synthesis. We characterized a cis-acting element that controls the production of the LMT1 RNAs. Since manipulation of this cis-acting element in its native position (the L-ProI region of replicase) was not possible because the mutations negatively affect replication, a region (5'TR) surrounding the putative termination sites (nt approximately 550 to 1000) was duplicated in the 3' end of a CTV replicon to allow characterization. The duplicated sequence continued to produce a 5'-terminal plus-strand sgRNA, here much larger ( approximately 11 kb), apparently by termination. Surprisingly, a new 3'-terminal sgRNA was observed from the duplicated 5'TR. A large 3'-terminal sgRNA resulting from the putative promoter activity of the native 5'TR was not observed, possibly because of the down-regulation of a promoter approximately 19 kb from the 3' terminus. However, we were able to observe a sgRNA produced from the native 5'TR of a small defective RNA, which placed the native 5'TR closer to the 3' terminus, demonstrating sgRNA promoter activity of the native 5'TR. Deletion mutagenesis mapped the promoter and the terminator activities of the 5'TR (in the 3' position in the CTV replicon) to a 57-nt region, which was folded by the MFOLD computer program into two stem-loops. Mutations in the putative stem-loop structures equally reduced or prevented production of both the 3'- and 5'-terminal sgRNAs. These mutations, when introduced in frame in the native 5'TR, similarly abolished the synthesis of the LMT1 RNAs and presumably the large 3'-terminal sgRNA while having no impact on replication, demonstrating that neither 5'- nor 3'-terminal sgRNA is necessary for replication of the replicon or full-length CTV in protoplasts. Differences between the 5'TR, which produced two plus-strand sgRNAs, and the cis-acting elements controlling the 3' open reading frames, which produced additional minus-strand sgRNAs corresponding to the 3'-terminal mRNAs, suggest that the different sgRNA controller elements had different origins in the modular evolution of closteroviruses.     

RNA 3'-terminal phosphate cyclase activity and RNA ligation in HeLa cell extract.

HeLa cell extract contains RNA ligase activity that converts linear polyribonucleotides to covalently closed circles. RNA substrates containing 2',3'-cyclic phosphate and 5'-hydroxyl termini are circularized by formation of a normal 3',5' phosphodiester bond. This activity differs from a previously described wheat germ RNA ligase which circularizes molecules with 2',3'-cyclic and 5' phosphate ends by a 2'-phosphomonester, 3',5'-phosphodiester linkage (Konarska et al., Nature 293, 112-116, 1981; Proc. Natl. Acad. Sci. USA 79, 1474-1478, 1982). The HeLa cell ligase can also utilize molecules with 3'-phosphate ends. However, in this case ligation is preceded by an ATP-dependent conversion of the 3'-terminal phosphate to the 2',3' cyclic form by a novel activity, RNA 3'-terminal phosphate cyclase. Both RNA ligase and RNA 3'-terminal phosphate cyclase activities are also present in extract of Xenopus oocyte nuclei, consistent with a role in RNA processing.     

DNase VII of human placenta. Mechanism studies

The mechanism of the human placental DNase VII, described previously (Hollis, G. F., and Grossman, L. (1981) J. Biol. Chem. 256, 8074-8079) has been investigated in further detail. The enzyme initiates exonucleolytic hydrolysis from the 3'-end of DNA in a nonprocessive, or distributive, manner, regardless of whether the carbohydrate moiety associated with the 3'-terminal nucleotide contains H or OH at its 2' and 3' positions. DNase VII does not have associated RNase H activity; however, it is capable of removing 3'-terminal ribonucleotides. The enzyme also can hydrolyze DNA containing a terminal nucleotide lacking a purine or pyrimidine as well as termini containing noncomplementary nucleotides. DNase VII activity is product-inhibited by deoxynucleoside 5'-monophosphates. From kinetic studies, the mononucleotide deoxyadenylic acid is a noncompetitive inhibitor with a Ki = 0.3 mM. The resemblance of DNase VII to the 3'----5' exonuclease activity of Escherichia coli DNA polymerase I and its possible role in excision repair and proofreading are discussed.     

Preparation of 2-azidoadenosine 3',5'-[5'-32P]bisphosphate for incorporation into transfer RNA. Photoaffinity labeling of Escherichia coli ribosomes

2-Azidoadenosine was synthesized from 2-chloroadenosine by sequential reaction with hydrazine and nitrous acid and then bisphosphorylated with pyrophosphoryl chloride to form 2-azidoadenosine 3',5'-bisphosphate. The bisphosphate was labeled in the 5'-position using the exchange reaction catalyzed by T4 polynucleotide kinase in the presence of [gamma-32P]ATP. Polynucleotide kinase from a T4 mutant which lacks 3'-phosphatase activity (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) was required to facilitate this reaction. 2-Azidoadenosine 3',5'-[5'-32P]bisphosphate can serve as an efficient donor in the T4 RNA ligase reaction and can replace the 3'-terminal adenosine of yeast tRNAPhe with little effect on the amino acid acceptor activity of the tRNA. In addition, we show that the modified tRNAPhe derivative can be photochemically cross-linked to the Escherichia coli ribosome.     

The thermodynamics of 3'-terminal pyrene and guanosine for the design of isoenergetic 2'-O-methyl-RNA-LNA chimeric oligonucleotide probes of RNA structure

To facilitate design of short isoenergetic hybridization probes for RNA, we report the influence of adding 5'- or 3'-terminal 2'-O-methylguanosine (GM), LNA-guanosine (GL), or 3'-terminal pyrene pseudo-nucleotide (PPN) on the thermodynamic stability of 2'-O-methyl-RNA/RNA (2'-O-Me-RNA/RNA) duplexes with sequences 5'CMGMGMCMAM/3'AAXGCCGUXAA, where X is A, C, G, or U. A 3'-terminal GM or GL added to the 2'-O-Me-RNA strand to form a G-A, G-G or G-U mismatch enhances thermodynamic stability (DeltaDeltaG degrees 37) of the 2'-O-Me-RNA/RNA duplexes on average by 0.7 and 1.5 kcal/mol, respectively. A 3'-terminal GM or GL in a GM-C or GL-C pair stabilizes the 2'-O-Me-RNA/RNA duplex by 2.6 and 3.4 kcal/mol, respectively. A 5'-terminal GM or GL in a G-A or G-G mismatch provided less stabilization in comparison with a 3'-terminal G-A or G-G mismatch, but more stabilization in a G-C or G-U pair. In contrast to guanosine derivatives, pyrene residue (P) as PPN at the 3'-terminal position enhances thermodynamic stability of the 2'-O-Me-RNA/RNA duplexes on average by 2.3 +/- 0.1 kcal/mol, relatively independent of the type of ribonucleotide placed in the opposite strand. The thermodynamic data can be applied to design 2'-O-Me-RNA/RNA duplexes with enhanced thermodynamic stability that is also sequence independent. This is useful for design of hybridization probes to interrogate RNA structure and/or expression by microarray and other methods.     

The Tetrahymena intervening sequence ribonucleic acid enzyme is a phosphotransferase and an acid phosphatase

A shortened form of the Tetrahymena intervening sequence (IVS) RNA acts as an enzyme, catalyzing nucleotidyl transfer and hydrolysis reactions with oligo(cytidylic acid) substrates [Zaug, A. J., & Cech, T. R. (1986) Science (Washington, D.C.) 231, 470-475]. These reactions involve phosphodiester substrates. We now show that the same enzyme has activity toward phosphate monoesters. The 3'-phosphate of C5p or C6p is transferred to the 3'-terminal guanosine of the enzyme. The pH dependence of the reaction (optimum at pH 5) indicates that the enzyme has activity toward the dianion and much greater activity toward the monoanion form of the 3'-phosphate of the substrate. Phosphorylation of the enzyme is reversible by C5-OH and other oligo(pyrimidines) such as UCU-OH. Thus, the RNA enzyme acts as a phosphotransferase, transferring the 3'-terminal phosphate of C5p to UCU-OH with multiple turnover. At pH 4 and 5, the phosphoenzyme undergoes slow hydrolysis to yield inorganic phosphate. Thus, the enzyme has acid phosphatase activity. The RNA enzyme dephosphorylates oligonucleotide substrates with high sequence specificity, which distinguishes it from known protein enzymes.     

The reaction mechanism of ribonuclease II and its interaction with nucleic acid secondary structures.

Ribonuclease II is a processive 3'- to 5'-exoribonuclease in Escherichia coli with two binding sites: a catalytic site associated with the first few 3'-nucleotides and an anchor site binding nucleotides approximately 15 to 25 from the 3'-end. When RNase II degrades single-stranded helical poly(C), the enzyme-substrate complex dissociates at discrete intervals of 12 nucleotides. RNase II stalled at the last rC of single-stranded 3'-(rC)(n)(dC)(m) oligonucleotides. The more residues released, the faster the stalled complex dissociated and the less it inhibited RNase II activity, i.e. the enzyme-substrate association weakened progressively. Using phosphodiesterase I (PDE I) as a probe, a method was developed to identify cytidine residues in (32)P-oligonucleotides interacting with a protein. PAGE bands corresponding to nucleotides 1-6 from the 3'-end were consistent with interaction at the catalytic site, and following a gap, bands approximately 15 to 25 from the 3'-end, with anchor site association. Both 3' and 5' binding were necessary to maintain the complex. Of most significance, the original anchor site nucleotides remained fixed at the anchor site while the 3'-end was pulled, or threaded, through the catalytic site, i.e. the substrate did not 'slide' through the enzyme. DNA oligonucleotides with double-stranded stem-loops were good competitive inhibitors of RNase II. A 3'-single-stranded arm was essential, while optimal binding required both 5'- and 3'-arms. PDE I probing indicated that the nucleotides at the anchor site were specified by the spatial distance from the catalytic site, and on only one of the duplex strands. When degradation of a structured RNA paused or stopped, the RNase II-product commenced cycles of dissociation-reassociation. Duplex strand binding by RNase II made complex DNA or RNA structures accessible to degradation by other nucleases and further verified the PDE I footprinting method.     

Modification of RNA by mRNA guanylyltransferase and mRNA (guanine-7-)methyltransferase from vaccinia virions.

A purified enzyme system isolated from vaccinia virus cores has been shown to modify the 5' termini of viral mRNA and synthetic poly(A) and poly(G) to form the structures m7G(5')pppA- and m7G(5')pppG-. The enzyme system has both guanylyltransferase and methyltransferase activities. The GTP:mRNA guanylyltransferase activity incorporates GMP into the 5' terminus via a 5'-5' triphosphate bond. The properties of this reaction are: (a) of the four nucleoside triphosphates only GTP is a donor, (b) mRNA with two phosphates at the 5' terminus is an acceptor while RNA with a single 5'-terminal phosphate is not, (c) Mg2+ is required, (d) the pH optimum is 7.8, (e) PP1 is a strong inhibitor, and (f) the reverse reaction, namely the formation of GTP from PP1 and RNA containing the 5'-terminal structure G(5')pppN-, readily occurs. The S-adenosylmethionine:mRNA(guanine-7-)methyltransferase activity catalyzes the methylation of the 5'-terminal guanosine. This reaction exhibits the following characteristics: (a) mRNA with the 5'-terminal sequences G(5')pppA- and G(5')pppG- are acceptors, (b) only position 7 of the terminal guanosine is methylated; internal or conventional 5'-terminal guanosine residues are not methylated, (c) the reaction is not dependent upon GTP or divalent cations, (d) optimal activity is observed in a broad pH range around neutrality, (e) the reaction is inhibited by S-adenosylhomocysteine. Both the guanylyltransferase and methyltransferase reactions exhibit bisubstrate kinetics and proceed via a sequential mechanism. The reactions may be summarized: (see article).     

Nucleoside triphosphate phosphohydrolase associated with cytoplasmic polyhedrosis virus.

Nucleoside triphosphate phosphohydrolase [EC 3.6.1.15] activity was found to be included in silkworm cytoplasmic polyhedrosis (CP) virus, which synthesizes mRNA carrying the 5'-terminal modification. This enzyme releases orthophosphate from the gamma-position in a nucleoside triphosphate, leaving nucleoside diphosphate. The rate of hydrolysis of ATP is faster than that of any other ribonucleoside triphosphate. Deoxy ATP is hydrolyzed rather faster than ATP. However, polynucleotides carrying triphosphate at the 5'-terminus, that is, 4S RNA which was synthesized by E. coli RNA polymerase [EC 2.7.7.6] using calf thymus DNA as a template, and the phage Q beta RNA (30S), are not effective substrates for this enzyme. Although the CP virion loses the viral genome and one kind of protein component on proteolytic treatment with pronase, the partially degraded virion still retains phosphohydrolase activity. The phosphohydrolase must therefore be associated firmly with the virion. This enzyme does not require the presence of nucleic acid for its function. Phosphohydrolysis of ATP by this enzyme activity represents a first step in the synthesis of the 5'-terminal modified mRNA of CP virus.     

Human 2'-phosphodiesterase localizes to the mitochondrial matrix with a putative function in mitochondrial RNA turnover

The vertebrate 2-5A system is part of the innate immune system and central to cellular antiviral defense. Upon activation by viral double-stranded RNA, 5'-triphosphorylated, 2'-5'-linked oligoadenylate polyribonucleotides (2-5As) are synthesized by one of several 2'-5'-oligoadenylate synthetases. These unusual oligonucleotides activate RNase L, an unspecific endoribonuclease that mediates viral and cellular RNA breakdown. Subsequently, the 2-5As are removed by a 2'-phosphodiesterase (2'-PDE), an enzyme that apart from breaking 2'-5' bonds also degrades regular, 3'-5'-linked oligoadenylates. Interestingly, 2'-PDE shares both functionally and structurally characteristics with the CCR4-type exonuclease-endonuclease-phosphatase family of deadenylases. Here we show that 2'-PDE locates to the mitochondrial matrix of human cells, and comprise an active 3'-5' exoribonuclease exhibiting a preference for oligo-adenosine RNA like canonical cytoplasmic deadenylases. Furthermore, we document a marked negative association between 2'-PDE and mitochondrial mRNA levels following siRNA-directed knockdown and plasmid-mediated overexpression, respectively. The results indicate that 2'-PDE, apart from playing a role in the cellular immune system, may also function in mitochondrial RNA turnover.     

Nucleotide sequence and stability of the RNA component of RNase P from a temperature-sensitive mutant of E. coli.

The gene coding for the RNA component of RNase P was cloned from a temperature-sensitive mutant of Escherichia coli defective in RNase P activity (ts709) and its parental wild-type strain (4273), and the complete nucleotide sequences of the gene and its flanking regions were determined. The 5'- and 3'-terminal sequences of the RNA component were determined and mapped on the DNA sequence. The mutant gene has GC-to-AT substitutions at positions corresponding to 89 and 365 nucleotides downstream from the 5' terminus of the RNA sequence. Comparing to the wild-type RNA, the mutant RNA is less stable and rapidly degraded in vivo and in vitro.     

Preparation and sequencing of the cloacin fragment of Streptomyces aureofaciens 16S RNA

A fast method for isolation of a 3'-terminal fragment of Streptomyces aureofaciens 16S RNA was developed. The procedure involves reaction of 70S ribosomes with cloacin DF13 and subsequent fractionation of the reaction mixture by polyacrylamide gel electrophoresis. The cloacin fragment was eluted from the gel and used directly for 3'-end labeling with cytidine-3',5'-[5'-32P]bisphosphate. The labeled RNA fragment was sequenced by the enzymatic method. It consists of 50 nucleotides and has the sequence 5'-GUCGUAACAAGGUAACCGUACCGGA-AGGUGCGGUUGGAUCACCUCCUUUCOH. The differences from the E. coli and Bacillus sequences and their possible influence on the rate and specificity of polypeptide synthesis are discussed.     

Zn2+-dependent deoxyribozymes that form natural and unnatural RNA linkages

We report Zn(2+)-dependent deoxyribozymes that ligate RNA. The DNA enzymes were identified by in vitro selection and ligate RNA with k(obs) up to 0.5 min(-)(1) at 1 mM Zn(2+) and 23 degrees C, pH 7.9, which is substantially faster than our previously reported Mg(2+)-dependent deoxyribozymes. Each new Zn(2+)-dependent deoxyribozyme mediates the reaction of a specific nucleophile on one RNA substrate with a 2',3'-cyclic phosphate on a second RNA substrate. Some of the Zn(2+)-dependent deoxyribozymes create native 3'-5' RNA linkages (with k(obs) up to 0.02 min(-)(1)), whereas all of our previous Mg(2+)-dependent deoxyribozymes that use a 2',3'-cyclic phosphate create non-native 2'-5' RNA linkages. On this basis, Zn(2+)-dependent deoxyribozymes have promise for synthesis of native 3'-5'-linked RNA using 2',3'-cyclic phosphate RNA substrates, although these particular Zn(2+)-dependent deoxyribozymes are likely not useful for this practical application. Some of the new Zn(2+)-dependent deoxyribozymes instead create non-native 2'-5' linkages, just like their Mg(2+) counterparts. Unexpectedly, other Zn(2+)-dependent deoxyribozymes synthesize one of three unnatural linkages that are formed upon the reaction of an RNA nucleophile other than a 5'-hydroxyl group. Two of these unnatural linkages are the 3'-2' and 2'-2' linear junctions created when the 2'-hydroxyl of the 5'-terminal guanosine of one RNA substrate attacks the 2',3'-cyclic phosphate of the second RNA substrate. The third unnatural linkage is a branched RNA that results from attack of a specific internal 2'-hydroxyl of one RNA substrate at the 2',3'-cyclic phosphate. When compared with the consistent creation of 2'-5' linkages by Mg(2+)-dependent ligation, formation of this variety of RNA ligation products by Zn(2+)-dependent deoxyribozymes highlights the versatility of transition metals such as Zn(2+) for mediating nucleic acid catalysis.     

Excision of 5'-terminal deoxyribose phosphate from damaged DNA is catalyzed by the Fpg protein of Escherichia coli.

Homogeneous Fpg protein of Escherichia coli has DNA glycosylase activity which excises some purine bases with damaged imidazole rings, and an activity excising deoxyribose (dR) from DNA at abasic (AP) sites leaving a gap bordered by 5'- and 3'-phosphoryl groups. In addition to these two reported activities, we show that the Fpg protein also catalyzes the excision of 5'-terminal deoxyribose phosphate (dRp) from DNA, which is the principal product formed by the incision of AP endonucleases at abasic sites. Moreover, the rate of the Fpg protein catalysis for the 2,6-diamino-4-hydroxy-5-formamidopyrimidine-DNA glycosylase activity is slower than the activities excising dR from abasic sites and dRp from abasic sites preincised by endonucleases. The product released by the Fpg protein in the excision of 5'-terminal dRp from an abasic site preincised by an AP endonuclease is a single base-free unsaturated dRp, suggesting that the excision results from beta-elimination. The release of 5'-terminal dRp by crude extracts of E. coli from wild type and fpg-mutant strains shows that the Fpg protein is one of the major EDTA-resistant activities catalyzing this reaction.     

Reovirus-induced Ribonucleic Acid Polymerase

A virus-induced ribonucleic acid (RNA) polymerase activity was found in L cells infected with type 3 reovirus. Most of the enzyme is associated with the "large particle" fraction of the infected cells. The enzyme first appeared at 3 to 5 hr after infection and increased in amount until 7 to 9 hr. All four ribonucleoside triphosphates are incorporated in vitro into an acid-insoluble form by the enzyme. The major part of the product formed in vitro is a double-stranded RNA indistinguishable from viral RNA by electrophoresis on polyacrylamide gel. Approximately 40% of the product is a single-stranded RNA of relatively small molecular weight. More than 95% of the nucleotides incorporated into double-stranded RNA by the enzyme are bound in internal 3'-5'-phosphodiester linkages extending back from both 3'- and 5'-termini of the RNA strands.