Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

As a starting point for the study of the biosynthesis of polyadenylated RNA in bacteria, the characteristics of RNA synthesis by cells of Escherichia coli B made permeable to small molecules by treatment with toluene were examined. Such cells mediated the incorporation of radiolabeled ribonucleoside triphosphates into RNA in a reaction that was sensitive to inhibitors of RNA polymerase and required the simultaneous presence of the four ribonucleoside triphosphates. Between 10 to 15% of the RNA synthesized under these conditions was polyadenylated as shown by affinity chromatography on oligo(dT)-cellulose. The presence of orthophosphate or dADP, inhibitors of polynucleotide phosphorylase, had no effect on the reaction and the rate of RNA synthesis was indistinguishable in the polynucleotide phosphorylase-deficient strain PR-7 and in its otherwise isogenic parent strain PR-100. The poly(A) tracts associated with the newly synthesized RNA could be isolated after exhaustive digestion with pancreatic and T1 ribonucleases and accounted for 14% of the poly(A)-RNA. At least 74% of the poly(A) sequences were located at the 3' ends of RNA molecules and their weight-average length was 48 nucleotide residues. The size distribution of total RNA and poly(A)-RNA synthesized in the toluenized cell system was similar to that of the corresponding pulse-labeled fractions derived from growing cultures. The sequence complexity of poly(A)-RNA and unadenylated RNA synthesized in toluenized cells with [alpha-32P]CTP as the labeled substrate was analyzed by hybridization to fragments of Escherichia coli B DNA generated by digestion with EcoRI restriction endonuclease and immobilized on nitrocellulose sheets. Both RNA fractions hybridized with many DNA fractions, the hybridization patterns being similar with poly(A)-RNA and unadenylated RNA. This indicated that many different types of RNA transcripts synthesized in toluenized cells were subject to polyadenylation, but that polyadenylation was incomplete so that each transcript was present in both an adenylated and an unadenylated state.
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PMID:Synthesis of polyadenylate-containing RNA in vitro in permeable cells of Escherichia coli B. 619 64

Synthesis of a complementary strand on the circular viral (+)-DNA of phage phiX174, coated with single-stranded DNA binding protein, is primed by the synthesis of an oligonucleotide by the primosome. Processive primosome movement on the lagging strand with the replication fork was proposed as a model for the discontinuous portion of Escherichia coli chromosome replication (Arai, K. and Kornberg, A. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 69-73; Arai, K., Low, R. L., and Kornberg, A. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 707-711). RNA primers covalently bound to the 5'-end of a DNA chain are heterogeneous with respect to both size and nucleotide composition. The chain length of the DNA-linked RNA primers is shorter than a decanucleotide, predominantly ranging from 1 to 9 residues. The primers start with adenylate followed mainly by a purine nucleotide (Pu) at the second position suggesting that pppA-Pu is a preferred initiation sequence. The inner sequences are more heterogeneous and no consensus or preferred sequence was found beyond the third position. The size distribution of the primer is influenced by the relative concentration of ribo- and deoxyribonucleoside triphosphates; the proportion of mononucleotide (riboadenylate) primer increases upon decreasing the relative ribonucleoside triphosphate concentration. Mapping of the transition sites from RNA to DNA on HaeIII endonuclease fragments suggest they are distributed randomly and occur frequently on the phiX174 genome. These results suggest that the selection of RNA priming sites is affected by primase at the preferred sequence 3'-T-pyrimidine nucleotide-5' on the template within the DNA domain generated by the dnaB protein. These properties of RNA priming have important implications for site selection by the primosome on the lagging strand at the replication fork of the E. coli chromosome.
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PMID:Site selection and structure of DNA-linked RNA primers synthesized by the primosome in phage phi X174 DNA replication in vitro. 619 63

We propose a mechanism for the priming of influenza viral RNA transcription by capped RNAs in which specific 5'-terminal fragments are cleaved from the capped RNAs by a virion-associated endonuclease. These fragments would serve as the actual primers for the initiation of transcription by the initial incorporation by the initial incorporation of a G residue at their 3' end. We show that virions and purified viral cores contain a unique endonuclease that cleaves RNAs containing a 5' methylated cap structure (m7GpppXm) preferentially at purine residues 10 to 14 nucleotides from the cap, generating fragments with 3'-terminal hydroxyl groups. RNAs containing the 5'-terminal structure GpppG could not be cleaved to produce these specific fragments. Consistent with our proposed mechanism, those capped fragments that function as primers could be linked to a G residue in transcriptase reactions containing alpha-32P-GTP as the only ribonucleoside triphosphate. The pattern of G and C incorporation onto these primer fragments suggests that this incorporation is directed by the second and third bases at the 3' end of the virion RNA template, which has the sequence 3' UCG. Primer fragments with a 3'-terminal A residue were used more efficiently than those with a 3'-terminal G residue, indicating a preference for generating an AGC sequence in the viral mRNA complementary to the 3' end of the virion RNA. Cleavage of the RNA primer and initiation of transcription are not necessarily coupled, because a 5' fragment isolated from one reaction could be used as a primer when added to a second reaction. Uncapped ribopolymer inhibitors of viral RNA transcription inhibited the cleavage of capped RNAs.
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PMID:A unique cap(m7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription. 626 60

The accessibility of extracellular and nuclear simian virus 40 (SV40-M and SV40-I, respectively) virion chromatin DNAs to micrococcal nuclease, DNase I, BglI, EcoRI, and RNA polymerase was examined. Our results support the following conclusions: (i) the intranucleosomal DNA of SV40-I chromatin, similar to the precursor 75S chromatin complex, is resistant to enzymatic activity; and (ii) SV40-M virion chromatin is modified in a manner which increases the accessibility of viral DNA to enzymes, and the distinction between nucleosomal DNA and linker DNA is absent. Micrococcal nuclease digestion of SV40-I virion chromatin gave a typical nucleosomal DNA ladder pattern with a repeat unit of 205 base pairs of DNA. SV40-I chromatin was sensitive to cleavage with endonuclease BglI, but not with EcoRI. When SV40-I virion chromatin was used as a template, the rate of incorporation of ribonucleoside triphosphates into RNA was 5% of that obtained with naked form SV40 form I DNA. Micrococcal nuclease digestion of SV40-M virion chromatin resulted in submonomeric DNA fragments of approximately 55 base pairs, but no larger repeating unit of DNA was observed. SV40-M virion chromatin was sensitive to cleavage with either BglI or EcoRI and was approximately 20% more susceptible to digestion with DNase I than was SV40-I virion chromatin. The transcriptional efficiency of the extracellular virion chromatin was almost equivalent to that of naked SV40 form I DNA and was 16-fold higher than the rate observed with nuclear virion chromatin. The increased transcriptional activity was dependent upon the presence of nonhistone viral protein VP1 or VP2 or both.
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PMID:Simian virus 40 maturation: chromatin modifications increase the accessibility of viral DNA to nuclease and RNA polymerase. 626 46

An endonuclease, which was originally identified for its RNA polymerase inhibitory activity, was isolated from rat liver endoplasmic reticulum. The enzyme yields on gel chromatography four active fractions of different molecular weights (Mr 5.3 X 10(4), 9 X 10(4), 1.55 X 10(5) and Sephacryl S-200 fraction at V0). Each fraction contains polypeptide chains which give a single band on sodium dodecylsulphate electrophoresis (Mr 5.4 X 10(4). This indicates that the enzyme is an oligomeric protein and each of its subunits exhibits the same or very similar molecular weights. Deoxyribonucleoside and ribonucleoside triphosphates can bind to the endoplasmic reticulum nuclease. Binding is enhanced in the presence of divalent cations particularly Mg2+. The enzyme exhibits mainly RNase activity but can also degrade denatured DNA and DNA . RNA hybrids which contain breaks in one of the two strands. Poly(A) and mainly poly(U) are most susceptible to its nucleolytic activity whereas poly(C) is completely resistant.
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PMID:Endoplasmic reticulum nuclease. Purification and specificity. 627 70

A crude replication complex prepared from enterovirus 70-infected cells was used to study the temperature-sensitive characteristic of the virus. The complex showed a temperature sensitivity in the in vitro incorporation of radiolabeled ribonucleoside triphosphate. The endonuclease itself did not account for the restricted RNA synthesis at the nonpermissive temperature. Analyses of the in vitro products by both gel electrophoresis and sucrose density gradient centrifugation showed that the complex synthesized three types of viral RNA only when incubated for a short period of time at the nonpermissive temperature. When the replication complex was treated with a detergent (deoxycholic acid), incorporation of ribonucleoside triphosphate into RNA at the permissive temperature was reduced to the level of that at the nonpermissive temperature. In addition, the in vitro RNA synthesis by the enterovirus 70 replication complex at the permissive temperature required a higher concentration of ATP than of other ribonucleoside triphosphates, whereas such a preference for ATP was not found in the reaction at the nonpermissive temperature. The results indicate that the initiation step of RNA synthesis by the complex is blocked at the nonpermissive temperature. The possible implications of these findings are discussed.
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PMID:Characterization of a temperature-sensitive defect of enterovirus 70: effect of elevated temperature on in vitro transcription. 632 38

Catalytic properties of the capped RNA-specific endonuclease associated with the influenza virus RNA polymerase were analyzed with use of synthetic hetero- and homopolymers containing 32P-labeled CAP structures at their 5' termini. The endonuclease displays its intrinsic activity provided that substrate RNA contains both the CAP-1 structure (m7GpppGm) and either A or U residues at 9 to 11 nucleotides distant from the CAP structure. Independent recognition of multiple RNA signals by the endonuclease was further supported by the findings that dinucleotide ApG, free CAP structures and RNA without the CAP structure inhibited the endonuclease activity to different extents. In the presence of four species of ribonucleoside 5'-triphosphates, the endonucleolytically cleaved fragments with the CAP-1 structure were incorporated into polynucleotides, supporting the concept that they are used as the primers for the transcription. The initial nucleotide linked to the primers was a G residue, the nucleotide complementary to the second base of the 3' termini of the vRNA segments.
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PMID:RNA polymerase of influenza virus. IV. Catalytic properties of the capped RNA endonuclease associated with the RNA polymerase. 685 61

Purified influenza viral cores catalyze the entire process of viral RNA transcription, which includes the endonucleolytic cleavage of heterologous RNAs containing cap 1 (m(7)GpppNm) structures to generate capped primers 10-13 nucleotides long, the initiation of transcription via the incorporation of a guanosine residue onto the primers, and elongation of the viral mRNAs [Plotch, S. J., Bouloy, M., Ulmanen, L & Krug, R. M. (1980) Cell 23, 847-858]. To identify which viral core protein (nucleocapsid protein, P1, P2, or P3) recognizes the cap 1 structure on the RNA primer, we irradiated (UV) endonuclease reactions carried out by viral cores in the absence of ribonucleoside triphosphates, with a primer RNA labeled in its cap 1 structure with (32)P. The labeled cap was crosslinked to a protein that had a mobility similar to that of the P3 protein, the smaller of the two basic P proteins, in both one- and two-dimensional gel electrophoresis. This strongly suggests that this crosslinked protein is the viral P3 protein. Competition experiments with unlabeled RNAs containing or lacking a cap 1 structure established that this protein recognizes the cap 1 structure on RNAs. This protein remained associated with the cap throughout the transcription reaction, even after the viral mRNA molecules were elongated. To identify the viral core protein that catalyzes the initiation of transcription via the incorporation of a guanosine residue onto primer fragments, we irradiated transcription reactions carried out by viral cores in the presence of [alpha-(32)P]GTP as the only ribonucleoside triphosphate with an unlabeled primer RNA. A labeled guanosine residue was crosslinked to a protein that had a mobility similar to that of the P1 protein, the larger of the two basic P proteins, in both one-and two-dimensional gel electrophoresis. The transcription reaction conditions required to bring this protein in close association with a labeled guanosine residue so that crosslinking could occur indicated that this association most likely occurred coincident with the guanosine residue's being incorporated onto the primer. These results suggest that the viral P1 protein catalyzes this incorporation and hence initiates transcription.
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PMID:Role of two of the influenza virus core P proteins in recognizing cap 1 structures (m7GpppNm) on RNAs and in initiating viral RNA transcription. 695 Mar 80

The bacteriophage lambda terminase is composed of two subunits, gpNu1 and gpA. In vitro, the holoenzyme is a site-specific endonuclease, helicase, ATPase, and can package lambda DNA into proheads. gpA possesses ATPase and helicase activities which are similar to those of the holoenzyme. Both terminase and gpA can hydrolyze a wide range of deoxyribo- and ribonucleoside triphosphates to inorganic phosphate and the corresponding diphosphate. Nucleoside diphosphates are not substrates for either protein. ATPase of both proteins is stimulated by double-stranded DNA. The ATPase of gpA is protein concentration-dependent, while that of terminase is not. Helicase activity of both proteins is not concentration-dependent, and requires a hydrolyzable triphosphate. ATP, dATP, and GTP supported helicase activity, while adenosine 5'-(beta, gamma-methylene)triphosphate, adenosine 5'-3-O-(thio)triphosphate, ADP, CTP, and UTP did not. The kinetic parameters of ATPase and helicase activities were similar for both proteins, but packaging with terminase was optimal only at a significantly higher level of ATP. Packaging was detectable at significant levels with CTP and UTP, but not with GTP. Packaging also differed from ATPase and helicase in the utilization of divalent metal cations and susceptibility to various inhibitors.
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PMID:The in vitro ATPases of bacteriophage lambda terminase and its large subunit, gene product A. The relationship with their DNA helicase and packaging activities. 817 94

The L-21 Sca I ribozyme derived from the group I intron of Tetrahymena thermophila pre-rRNA catalyzes an endonuclease reaction analogous to the first step of self-splicing. Guanosine (G) is bound by the ribozyme, and its 3'-hydroxyl group acts as the nucleophile. Here, we provide evidence that Km for G in several single-turnover reactions is equal to the equilibrium dissociation constant for G. This evidence includes the observation that removal of the 2'-hydroxyl group at the cleavage site of the oligoribonucleotide substrate [from CCCUCUA to CCCUC(dU)A] decreases the rate of cleavage approximately 1000-fold but has no effect on either the Km for G (0.17 mM) or for guanosine 5'-monophosphate (pG) (0.09 mM). In the course of this study, it was observed that Km for G or pG was lower by a factor of 5 for reactions with the ribozyme-CCCUC(dU)A complex compared with the free ribozyme, indicating a modest amount of thermodynamic coupled binding of the two substrates. The decrease in the rate of oligonucleotide dissociation upon addition of saturating pG provides independent support for this coupling. Coupling is lost with a substrate that cannot make the normal tertiary interactions with the ribozyme, providing evidence that coupled binding requires docking of the substrate into the catalytic core. Surprisingly, the binding of product CCCUCU and G is slightly anticooperative, indicating that the cleaved pA is important for coupling with substrate. Coupled binding suggests a splicing model in which the intron binds G tightly to promote the first step of reaction, after which its binding is an order of magnitude weaker, thereby facilitating the second step.
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PMID:Guanosine binding to the Tetrahymena ribozyme: thermodynamic coupling with oligonucleotide binding. 837 6


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