Purification and Properties of Bacteriophage T4-Induced RNA Ligase

An enzyme, purified 300-fold from Escherichia coli infected with bacteriophage T4, catalyzes the conversion of 5'-termini of polyribonucleotides to internal phosphodiester bonds. The reaction requires ATP and Mg(++). For every 5'-(32)P terminus rendered resistant to alkaline phosphatase, an equal amount of AMP and PPi are formed. Various polyribonucleotides are substrates in the reaction; to date, the best substrate is [5'-(32)P]polyriboadenylate. With the latter substrate, no evidence of intermolecular reaction was obtained. However, the 5'-(32)P termini of poly(A) rendered resistant to alkaline phosphatase are also resistant to attack by RNase II, polynucleotide phosphorylase, and low concentrations of venom phosphodiesterase. Since the product formed with poly(A) lacks 3'-hydroxyl ends, as measured with these exonucleases, the enzyme appears to convert linear molecules of polyriboadenylate to a circular form by the intramolecular covalent linkage of the 5'-phosphate end to the 3'-hydroxyl terminus.     

Isolation and characterization of an RNA ligase from HeLa cells.

An RNA ligase has been purified from HeLa cells, which catalyzes the intra- and intermolecular ligation of linear RNA substrates possessing 5'-hydroxyl and 2',3'-cyclic phosphate termini in the presence of ATP or dATP. In this reaction, the 2',3'-cyclic phosphate is incorporated into a 3'-5'-phosphodiester bond, in agreement with the findings of Filipowicz et al. [Filipowicz, W., Konarska, M., Gross, H. J. & Shatkin, A. J. (1983) Nucleic Acids Res. 11, 1405-1418]. The activity of the purified enzyme is dependent on the addition of ATP or dATP, a divalent cation (Mg2+), and 5'-hydroxyl, 2',3'-cyclic phosphate-terminated RNA substrates. No ligation occurs with the substrates OH(Up)10G(3')p or OH(Up)10G(2')p or with 5'-phosphate, 2',3'-cyclic phosphate-terminated oligoribonucleotides.     

Polyadenylic Acid at the 3′-Terminus of Poliovirus RNA

Poliovirus RNA that has been derivatized at the 3'-end with NaIO(4)-NaB(3)H(4) yields, after hydrolysis with alkali or RNase T2, predominantly labeled residues of modified adenosine; no labeled nucleoside derivative is produced by digestion with RNase A or RNase T1. The 3'-terminal bases of the RNA are, therefore,...ApA(OH). Hydrolyzates of poliovirus [(32)P]RNA, after exhaustive digestion with RNase T1 or RNase A, contain, besides internal oligonucleotides, polynucleotides resistant to further action of ribonucleases T1 and A, respectively; these polynucleotides were isolated by membrane-filter binding or ion-exchange chromatography. The sequence of the T1-resistant polynucleotide was determined to be (Ap)(n)A(OH), that of the RNase A-resistant polynucleotide was GpGp(Ap)(n)A(OH). The chain length (n) of the polyadenylic acid, as analyzed by different methods, averages 89 nucleotides. Gel electrophoresis revealed heterogeneity of the size of poly(A). Poliovirus RNA, when labeled in vitro at the 3'-end, contains [3'-(3)H]poly(A); when labeled in vivo with [(3)H]A, it contains [(3)H](Ap)(n)A(OH). The data establish that... YpGpGp(Ap)([unk])A(OH) is the 3'-terminal sequence of poliovirus RNA, Type 1 (Mahoney). Since this mammalian virus reproduces in the cell cytoplasm, these observations may modify prior interpretations of the function of polyadenylate ends on messenger RNAs.     

Isolation and characterization of RNA ligase from wheat germ.

A RNA ligase from wheat germ has been extensively purified. In the presence of ATP these enzyme preparations catalyze the covalent linkage of 5'-phosphate and 2',3'-cyclic phosphate termini of RNA chains. Concomitant with the formation of a 3',5'-phosphodiester linkage, the 2',3'-cyclic phosphate is converted to a 2'-phosphate ester, in accord with the findings of Konarska et al. [Konarska, M., Filipowicz, W. & Gross, H. J. (1982) Proc. Natl. Acad. Sci. USA 79, 1474-1478]. The action of the purified enzyme is totally dependent on ATP and on RNA substrates containing a 5'-phosphate terminus at one end and either a 2',3'-cyclic phosphate or a 2'-phosphate terminus at the other end. In the latter case, the reaction is about 30% as active as with the cyclic derivative. In contrast, RNA chains containing 3'-phosphate ends are less than 5% as active as those with the cyclic ends. Purified preparations of RNA ligase have an intrinsic ability to hydrolyze 2',3'-cyclic phosphate termini to 2'-phosphate termini. This reaction is readily detectable in the absence of ATP.     

T4-induced RNA ligase joins single-stranded oligoribonucleotides.

RNA ligase isolated from Escherichia coli infected with bacteriophage T4 will catalyze the formation of an intermolecular 3' leads to 5' phosphodiester linkage between an oligoribonucleotide with a free 3'-hydroxyl and another oligoribonucleotide with a 5'-phosphate. Upon reaction with (Ap)5C, nearly quantitative conversion of the hexamer [5'-32P]p(Up)5U to the dodecamer (Ap)5C[3' leads to 5'-32P]p(Up)5U was observed. The product was identified by its mobility on RPC-5 column chromatography, its resistance to alkaline phosphatase, and the appearance of the expected radiolabeled products on hydrolysis with alkali, ribonuclease A, snake venom phosphodiesterase, and spleen phosphodiesterase. The coupling of other pairs of single-stranded oligoribonucleotides has also been demonstrated. The intermolecular joining reaction is probably mechanistically similar to the intramolecular cyclization activity previously reported for Tr RNA ligase. It is expected that this enzyme will be useful for the synthesis of RNA fragments of defined sequence.     

An immunological determinant of RNase P protein is conserved between Escherichia coli and humans.

RNase P, an enzyme with RNA and protein subunits, cleaves tRNA precursor molecules to form the 5' termini of mature tRNAs in both prokaryotes and eukaryotes. Rabbit antibodies made against the protein subunit, C5 protein, of Escherichia coli RNase P bound RNase P protein from E. coli and Bacillus subtilis in immunoblots and solid-phase immunoassays. These rabbit anti-C5 antibodies also bound a protein (Mr approximately 40,000) in preparations of RNase P from human (HeLa) cells and depleted the enzymatic activity from preparations of RNase P from both human and E. coli cells. Finally, rabbit anti-C5 antibodies immunoprecipitated from crude extracts of human cells a ribonucleoprotein complex containing H1 RNA, the putative RNA component of human RNase P. These results show that an antigenic determinant is shared by C5 protein from E. coli RNase P and a protein component of RNase P from human cells.     

The catalytic domain of RNase E shows inherent 3' to 5' directionality in cleavage site selection

RNase E, a multifunctional endoribonuclease of Escherichia coli, attacks substrates at highly specific sites. By using synthetic oligoribonucleotides containing repeats of identical target sequences protected from cleavage by 2'-O-methylated nucleotide substitutions at specific positions, we investigated how RNase E identifies its cleavage sites. We found that the RNase E catalytic domain (i.e., N-Rne) binds selectively to 5'-monophosphate RNA termini but has an inherent mode of cleavage in the 3' to 5' direction. Target sequences made uncleavable by the introduction of 2'-O-methyl-modified nucleotides bind to RNase E and impede cleavages at normally susceptible sites located 5' to, but not 3' to, the protected target. Our results indicate that RNase E can identify cleavage sites by a 3' to 5' "scanning" mechanism and imply that anchoring of the enzyme to the 5'-monophosphorylated end of these substrates orients the enzyme for directional cleavages that occur in a processive or quasiprocessive mode. In contrast, we find that RNase G, which has extensive structural homology with and size similarity to N-Rne, and can functionally complement RNase E gene deletions when overexpressed, has a nondirectional and distributive mode of action.     

MECHANISM OF DNA CHAIN GROWTH, IV. DIRECTION OF SYNTHESIS OF T4 SHORT DNA CHAINS AS REVEALED BY EXONUCLEOLYTIC DEGRADATION

T4 nascent short chains labeled at their growing ends with H(3)-thymidine and uniformly with C(14)-thymidine were prepared, separated into complementary strands, and degraded by E. coli exonuclease I in the 3' to 5' direction or by B. subtilis nuclease in the 5' to 3' direction. The kinetics of release of H(3) and C(14) labels by both enzymes was consistent with the conclusion that the H(3) label is at the 3' end of the nascent short chains of both strands and that the short chains are products of discontinuous synthesis in the 5' to 3' direction along the two template strands.     

Altered degradation of circulating nucleic acids and oligonucleotides in diabetic patients

Foreign, infection-associated or endogenously generated circulating nucleotide motifs may represent the critical determinants for the activation of the Toll-like receptors (TLRs), leading to immune stimulation and cytokine secretion. The importance of circulating nucleases is to destroy nucleic acids and oligonucleotides in the blood stream and during cell entry. Patients with juvenile insulin-dependent diabetes, adult patients with insulin-dependent diabetes and adult patients with type 2 diabetes were allocated to the study, together with the age-matched control subjects. Plasma RNase and nuclease activity were examined, in relation to different substrates-TLRs response modifiers, and circulating RNA and oligonucleotides were isolated. The fall in enzyme activity in plasma was obtained for rRNA, poly(C), poly(U), poly(I:C), poly(A:U) and CpG, especially in juvenile diabetics. In order to test the non-enzymatic glycation, commercial RNase (E.C.3.1.27.5) and control plasma samples were incubated with increasing glucose concentrations (5, 10, 20 and 50 mmol/l). The fall of enzyme activity was expressed more significantly in control plasma samples than for the commercial enzyme. Total amount of purified plasma RNA and oligonucleotides was significantly higher in diabetic patients, especially in juvenile diabetics. The increase in the concentration of nucleotides corresponded to the peak absorbance at 270 nm, similar to polyC. The electrophoretic bands shared similar characteristics between controls and each type of diabetic patients, except that the bands were more expressed in diabetic patients. Decreased RNase activity and related increase of circulating oligonucleotides may favor the increase of nucleic acid "danger motifs", leading to TLRs activation.     

The Homopolyadenylate and Adjacent Nucleotides at the 3′-Terminus of 30-40S RNA Subunits in the Genome of Murine Sarcoma-Leukemia Virus

Adenosine is the major 3'OH-terminal nucleoside of the 60-70S RNA genome of the murine sarcoma-leukemia virus, its 30-40S RNA subunits, and the poly(A) segments derived by RNase treatment of both RNA species, as determined by periodate oxidation-[(3)H]-borohydride reduction. The binding 30-40S RNA to oligo(dT)-cellulose suggests that most viral RNA subunits contain poly(A). The molecular weight of poly(A) derived from viral RNA by digestion with RNase and purified by affinity chromatography is 64,000-68,000, as determined by gel electrophoresis. From the size of poly(A) and the poly(A) content of viral RNA (1.6%), it is estimated that there is about one poly(A) segment for each viral 30-40S RNA subunit. The results of 3'-termini labeling with [(3)H]borohydride, in vivo labeling with [(3)H]adenosine, and base composition of [(32)P]poly(A) indicate that a homopoly(A) segment is located at the 3'-end of a 30-40S RNA subunit. The homogeneous poly(A) segments isolated from RNase T1 digests of 60-70S [(32)P]RNA consist of one cytidylate, one uridylate, and about 190 adenylate residues, while those isolated from RNase A digests consist exclusively of adenylate residues. These results indicate that -G(C,U)A(190)A(OH) is the 3'-terminal nucleotide sequence of the viral 30-40S RNA subunits.     

Isolation and characterization of a second RNase H (RNase HII) of Escherichia coli K-12 encoded by the rnhB gene.

An additional RNase H (EC 3.1.26.4), RNase HII, has been isolated from Escherichia coli K-12. By screening a library of E. coli DNA for clones that suppressed RNase H deficiency of an E. coli rnh mutant, a clone was obtained that produced a protein with RNase H activity. The overexpressed RNase HIII protein in E. coli was purified to near homogeneity and exhibited a strong preference for the ribonucleotide moiety of RNA-DNA hybrid as substrate. The terminal 11 amino acids were determined and were identical to those predicted from the nucleotide sequence. The rnhB gene, which encodes RNase HII, was distinct from rnhA by its map position (4.5 min on E. coli genetic map, between lpxB and dnaE) and by the lack of significant amino acid sequence similarity. The presence of a second RNase H in E. coli indicates that multiple RNase H genes per genome is a general feature of a general feature of a wide variety of organisms.     

The tRNA processing enzyme RNase T is essential for maturation of 5S RNA.

The maturation of 5S RNA in Escherichia coli is poorly understood. Although it is known that large precursors of 5S RNA accumulate in mutant cells lacking the endoribonuclease-RNase E, almost nothing is known about how the mature 5' and 3' termini of these molecules are generated. We have examined 5S RNA maturation in wild-type and single- or multiple-exoribonuclease-deficient cells by Northern blot and primer-extension analysis. Our results indicate that no mature 5S RNA is made in RNase T-deficient strains. Rather, 5S RNA precursors containing predominantly 2 extra nucleotides at the 3' end accumulate. Apparently, these 5S RNAs are functional inasmuch as mutant cells are viable, growing only slightly slower than wild type. Purified RNase T can remove the extra 3' residues, showing that it is directly involved in the trimming reaction. In contrast, mutations affecting other 3' exoribonucleases have no effect on 5S RNA maturation. Approximately 90% of the 5S RNAs in both wild-type and RNase T- cells contain mature 5' termini, indicating that 5' processing is independent of RNase T action. These data identify the enzyme responsible for generating the mature 3' terminus of 5S RNA molecules and also demonstrate that a completely processed 5S RNA molecule is not essential for cell survival.     

Excision of misincorporated ribonucleotides in DNA by RNase H (type 2) and FEN-1 in cell-free extracts

Misincorporated ribonucleotides in DNA will cause DNA backbone distortion and may be targeted by DNA repair enzymes. Using double-stranded oligonucleotide probes containing a single ribose, we demonstrate a robust activity in human, yeast, and Escherichia coli cell-free extracts that nicks 5' of the ribose. The human and yeast extracts also make a subsequent cut 3' of the ribonucleotide releasing a ribonucleotide monophosphate. The resulting 1-nt gap is an ideal substrate for polymerase and ligase to complete a proposed repair sequence that effectively replaces the ribose with deoxyribose. Screening of yeast deletion mutant cells reveals that the initial nick is made by RNase H(35), a RNase H type 2 enzyme, and the second cut is made by Rad27p, the yeast homologue of human FEN-1 protein. RNase H type 2 enzymes are present in all kingdoms of life and are evolutionarily well conserved. We knocked out the corresponding rnhb gene in E. coli and show that extracts from this strain lack the nicking activity. Conversely, a highly purified archaeal RNase HII type 2 protein has a pronounced activity. To study substrate specificity, extracts were made from a yeast double mutant lacking the other main RNase H enzymes [RNase H1 and RNase H(70)], while maintaining RNase H(35). It was found that a single ribose is preferred as substrate over a stretch of riboses, further strengthening a proposed role of this enzyme in the repair of misincorporated ribonucleotides rather than (or in addition to) processing RNADNA hybrid molecules.     

Presence of a putative 15S precursor to β-globin mRNA but not to α-globin mRNA in Friend cells

Dimethyl sulfoxide-induced Friend cells were labeled for periods of 5-60 min. The denatured RNA was fractionated by sucrose gradient centrifugation and the distribution of alpha- and beta-globin-specific [(3)H]RNA was determined by hybridization to hybrid plasmids containing mouse alpha- and beta-globin DNA, respectively. After 5 min of labeling, a 15S peak of beta-globin-specific (but not alpha-globin-specific) [(3)H]RNA was detected, next to an equal amount of 10S beta-globin [(3)H]RNA. With increasing periods of labeling, the amount of 15S beta-globin [(3)H]RNA remained constant but the amount 10S beta-globin [(3)H]RNA increased steadily. alpha-Globin-specific [(3)H]RNA sedimented at 11 S after 5 min of labeling and at 9.5 S after longer labeling periods. Analysis of 15S globin-specific [(3)H]RNA purified by the poly(dC)-cDNA method [Curtis, P. J. & Weissmann, C. (1976) J. Mol. Biol. 106, 1061-1075] showed oligonucleotides characteristic of beta-globin mRNA but not of alpha-globin mRNA, as well as about 20 new oligonucleotides. Our results suggest that 10S beta-globin mRNA arises via a 15S precursor that has a half-life of 5 min or less; 9.5S alpha-globin mRNA may be derived from an 11S precursor.     

Catalytic activation of multimeric RNase E and RNase G by 5'-monophosphorylated RNA

RNase E is an endonuclease that plays a central role in RNA processing and degradation in Escherichia coli. Like its E. coli homolog RNase G, RNase E shows a marked preference for cleaving RNAs that bear a monophosphate, rather than a triphosphate or hydroxyl, at the 5' end. To investigate the mechanism by which 5'-terminal phosphorylation can influence distant cleavage events, we have developed fluorogenic RNA substrates that allow the activity of RNase E and RNase G to be quantified much more accurately and easily than before. Kinetic analysis of the cleavage of these substrates by RNase E and RNase G has revealed that 5' monophosphorylation accelerates the reaction not by improving substrate binding, but rather by enhancing the catalytic potency of these ribonucleases. Furthermore, the presence of a 5' monophosphate can increase the specificity of cleavage site selection within an RNA. Although monomeric forms of RNase E and RNase G can cut RNA, the ability of these enzymes to discriminate between RNA substrates on the basis of their 5' phosphorylation state requires the formation of protein multimers. Among the molecular mechanisms that could account for these properties are those in which 5'-end binding by one enzyme subunit induces a protein structural change that accelerates RNA cleavage by another subunit.