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1.
The role of non-classical hydrogen bonding in RNA structure has been investigated using polyuridylic acid, which has a labile ordered structure at temperatures near 0 °C, as a model system. By comparing the proton nuclear magnetic resonance spectrum of poly(U) in the transition region with that of uridine and the dimer UpU we find evidence that both the imino N(3)-H and the ribosyl 2′-OH protons are hydrogen bonded. The characteristics of the former are consistent with participation in N(3)-HOC bonding primarily between residues in the same strand. As yet we cannot unambiguously assign the acceptor for the 2′-OH in ordered poly(U): because of its apparent stability and the acceptable stereochemistry, we presently favor a bond between ribose 2′-OH and O(1′) connecting adjacent nucleotides of the same strand. This arrangement could contribute to the co-operativity of the poly(U) helix formation. The recently proposed 2′-OHO(1′) interactions in crystalline yeast transfer RNAPhe suggest similar interactions might play a role in the conformational stability of natural RNAs. A second conformational transition below the major transition in the ultraviolet can be detected in poly(U) by monitoring the H(6) proton of uracil.  相似文献   

2.
Hen1 is an RNA ribose 2′-O-methyltransferase that modifies the 3′ terminal nucleoside of eukaryal small regulatory RNAs. Here, we report that Hen1 homologs are present in bacterial proteomes from eight different phyla. Bacterial Hen1 is encoded by the proximal ORF of a two-gene operon that also encodes polynucleotide kinase-phosphatase (Pnkp), an RNA repair enzyme. Purified recombinant Clostridium thermocellum Hen1 is a homodimer of a 465-amino acid polypeptide. CthHen1 catalyzes methyl transfer from AdoMet to the 3′ terminal nucleoside of an RNA oligonucleotide, but is unreactive with a synonymous DNA oligonucleotide or an RNA with a single 3′-terminal deoxyribose sugar. CthHen1 is optimally active at alkaline pH and dependent on manganese. Activity is inhibited by AdoHcy and abolished by mutations D291A and D316A in the putative AdoMet-binding pocket. The C-terminal fragment, Hen1-(259–465), comprises an autonomous monomeric methyltransferase domain.  相似文献   

3.
4.
The RNA world hypothesis describes a stage in the early evolution of life in which RNA served as genome and as the only genome-encoded catalyst. To test whether RNA world organisms could have used cyclic trimetaphosphate as an energy source, we developed an in vitro selection strategy for isolating ribozymes that catalyze the triphosphorylation of RNA 5′-hydroxyl groups with trimetaphosphate. Several active sequences were isolated, and one ribozyme was analyzed in more detail. The ribozyme was truncated to 96 nt, while retaining full activity. It was converted to a trans-format and reacted with rates of 0.16 min−1 under optimal conditions. The secondary structure appears to contain a four-helical junction motif. This study showed that ribozymes can use trimetaphosphate to triphosphorylate RNA 5′-hydroxyl groups and suggested that RNA world organisms could have used trimetaphosphate as their energy source.  相似文献   

5.
The 3′ terminal sequences of RNAs 1, 2, 3 and 4 from each of the three bromoviruses (brome mosaic, cowpea chlorotic mottle and broad bean mottle viruses) and also from cucumber mosaic virus display interviral sequence similarity in addition to strong intraviral homology. Interviral similarity is much more evident when RNA secondary, rather than primary, structures are compared. The last 190 bases of the various RNAs can fold into strikingly similar, extensively base-paired secondary structures whose common features are supported by RNA structure mapping. The extreme 3′ end of each viral RNA can base-pair in two distinct configurations. Bromovirus RNA 3s each contain an unusually accessible internal oligo(A) sequence which, in brome mosaic virus at least, is located in the intercistronic noncoding region. Functional implications of these structural features are discussed.  相似文献   

6.
3′-deoxyadenosine triphosphate inhibited invitro [3H]UMP incorporation by RNA-dependent RNA polymerases from tobacco and cowpea plants. The inhibition of [3H]UMP incorporation could be reversed by simultaneous addition of higher ATP concentrations but not with increasing concentrations of UTP or when excess ATP was added 10 min after the inhibitor. These results suggest 3′-deoxyadenosine triphosphate competes specifically with ATP in reaction mixtures and results in premature termination of RNA synthesis invitro by RNA-dependent RNA polymerase.  相似文献   

7.
The influence of hypothalamic and pituitary type II 5′ deiodinase (5′D-II) activities and T3 content on pituitary TSH content was investigated in streptozotocin (STZ)-induced diabetic rats (D). The results show, first, that hypothalamic and pituitary 5′D-II activities were lower in neonatal D rats versus control (C) rats, and the normal developmental pattern was altered. Secondly, when D and C rats were thyroidectomized (Tx) at 25 days of age (D+Tx, C+Tx), pituitary and hypothalamic 5′D-II activities increased ten days later in both populationsvs. intact rats, but the percentage of increase was smaller in D+Tx than in C+Tx. The hypothalamic T3 to T4 ratios were also decreased in D+Tx animals (0.38) as compared to C+Tx rats (1.64). The hypothalamic T3 content was reduced by 30% in D as compared to C rats and by 80% in D+Tx as compared to C+Tx rats, showing a defect in hypothalamic T4 deiodination. Pituitary TSH content increased after Tx in D+Tx, but not in C+Tx. These results in diabetic rats indicate that the hypothalamic and pituitary 5′D-II activity and hypothalamic T3 content are affected by diabetes and play a role in the regulation of pituitary TSH content.  相似文献   

8.
Hausmann NZ  Znosko BM 《Biochemistry》2012,51(26):5359-5368
To better elucidate RNA structure-function relationships and to improve the design of pharmaceutical agents that target specific RNA motifs, an understanding of RNA primary, secondary, and tertiary structure is necessary. The prediction of RNA secondary structure from sequence is an intermediate step in predicting RNA three-dimensional structure. RNA secondary structure is typically predicted using a nearest neighbor model based on free energy parameters. The current free energy parameters for 2 × 3 nucleotide loops are based on a 23-member data set of 2 × 3 loops and internal loops of other sizes. A database of representative RNA secondary structures was searched to identify 2 × 3 nucleotide loops that occur in nature. Seventeen of the most frequent 2 × 3 nucleotide loops in this database were studied by optical melting experiments. Fifteen of these loops melted in a two-state manner, and the associated experimental ΔG°(37,2×3) values are, on average, 0.6 and 0.7 kcal/mol different from the values predicted for these internal loops using the predictive models proposed by Lu, Turner, and Mathews [Lu, Z. J., Turner, D. H., and Mathews, D. H. (2006) Nucleic Acids Res. 34, 4912-4924] and Chen and Turner [Chen, G., and Turner, D. H. (2006) Biochemistry 45, 4025-4043], respectively. These new ΔG°(37,2×3) values can be used to update the current algorithms that predict secondary structure from sequence. To improve free energy calculations for duplexes containing 2 × 3 nucleotide loops that still do not have experimentally determined free energy contributions, an updated predictive model was derived. This new model resulted from a linear regression analysis of the data reported here combined with 31 previously studied 2 × 3 nucleotide internal loops. Most of the values for the parameters in this new predictive model are within experimental error of those of the previous models, suggesting that approximations and assumptions associated with the derivation of the previous nearest neighbor parameters were valid. The updated predictive model predicts free energies of 2 × 3 nucleotide internal loops within 0.4 kcal/mol, on average, of the experimental free energy values. Both the experimental values and the updated predictive model can be used to improve secondary structure prediction from sequence.  相似文献   

9.
2′,3′-Cyclic nucleotide 3′-phosphohydrolase has been demonstrated in human erythrocyte membranes. The properties of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in erythrocyte membranes were similar to those of the brain enzyme with respect to pH optimum, substrate specificity and effects of metal ions. The estimation of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in various stages of membrane preparation showed that the enzyme in erythrocytes is exclusively associated with the membranes.  相似文献   

10.
Summary 2-Amino-2-deoxyuridine reacts efficiently with nucleoside 5-phosphorimidazolides in aqueous solution. The dinucleoside monophosphate analogues were obtained in yields exceeding 80% under conditions in which little reaction occurs with the natural nucleosides.In a similar way, the 5-phosphorimidazolide of 2-amino-2-deoxyuridine undergoes self-condensation in aqueous solution to give a complex mixture of oligomers.The phosphoramidate bond in the dinucleoside monophosphate analogues is stable for several days at room temperature and pH 7. The mechanisms of their hydrolysis under acidic and alkaline conditions are described.Abbreviations A adenosine - C cytidine - G guanosine - U uridine - T thymidine - UN3 2-azido-2-deoxyuridine - UNH2 2-amino-2-deoxyuridine - ImpA adenosine 5-phosphorimidazolide - ImpU uridine 5-phosphorimidazolide - ImpUN3 2-azido-2-deoxyuridine 5-phosphorimidazolide - ImpUNH2 2-amino-2-deoxyuridine 5-phosphorimidazolide - pA adenosine 5-phosphate - pU uridine 5-phosphate - pUN3 2-azido-2-deoxyuridine 5-phosphate - pUNH2 2-amino-2-deoxyuridine 5-phosphate - UpA uridylyl-[35]-adenosine - UpU uridylyl-[35]-uridine - UNpA adenylyl-[52]-2-amino-2-deoxy-uridine - UNpU uridylyl-[52]-2-amino-2-deoxyuridine (pUN)n n=2,3,4 [25]-linked oligomers of pUNH2 poly(A) polyadenylic acid - Im imidazole - MeIm l-methylimidazole  相似文献   

11.
Methylation of riboses at 2′-OH group is one of the most common RNA modifications found in number of cellular RNAs from almost any species which belong to all three life domains. This modification was extensively studied for decades in rRNAs and tRNAs, but recent data revealed the presence of 2′-O-methyl groups also in low abundant RNAs, like mRNAs.Ribose methylation is formed in RNA by two alternative enzymatic mechanisms: either by stand-alone protein enzymes or by complex assembly of proteins associated with snoRNA guides (sno(s)RNPs). In that case one catalytic subunit acts at various RNA sites, the specificity is provided by base pairing of the sno(s)RNA guide with the target RNA. In this review we compile available information on 2′-OH ribose methylation in different RNAs, enzymatic machineries involved in their biosynthesis and dynamics, as well as on the physiological functions of these modified residues.  相似文献   

12.
The misincorporation of 2′-deoxyribonucleotides (dNs) into RNA has important implications for the function of non-coding RNAs, the translational fidelity of coding RNAs and the mutagenic evolution of viral RNA genomes. However, quantitative appreciation for the degree to which dN misincorporation occurs is limited by the lack of analytical tools. Here, we report a method to hydrolyze RNA to release 2′-deoxyribonucleotide-ribonucleotide pairs (dNrN) that are then quantified by chromatography-coupled mass spectrometry (LC-MS). Using this platform, we found misincorporated dNs occurring at 1 per 103 to 105 ribonucleotide (nt) in mRNA, rRNAs and tRNA in human cells, Escherichia coli, Saccharomyces cerevisiae and, most abundantly, in the RNA genome of dengue virus. The frequency of dNs varied widely among organisms and sequence contexts, and partly reflected the in vitro discrimination efficiencies of different RNA polymerases against 2′-deoxyribonucleoside 5′-triphosphates (dNTPs). Further, we demonstrate a strong link between dN frequencies in RNA and the balance of dNTPs and ribonucleoside 5′-triphosphates (rNTPs) in the cellular pool, with significant stress-induced variation of dN incorporation. Potential implications of dNs in RNA are discussed, including the possibilities of dN incorporation in RNA as a contributing factor in viral evolution and human disease, and as a host immune defense mechanism against viral infections.  相似文献   

13.
2′,3′-Cyclic nucleotide 3′-phosphohydrolase (nucleoside-2′:3′-cyclic-phosphate 2′-nucleotidohydrolase, EC 3.1.4.37) activity has been demonstrated in rat liver mitochondria. The enzyme was localized in both the outer and inner mitochondrial membranes but was absent from the intermembrane space and matrix. The mitochondrial (cyclic nucleotide) phosphohydrolase was activated by freezing and thawing and by treatment with digitonin or detergents. It is suggested that (cyclic nucleotide) phosphohydrolase is an integral membrane protein which is buried to a significant degree within the membrane. Atractyloside was found to be a noncompetitive inhibitor of the enzyme both in intact mitochondria and in preparations of the mitochondrial membranes. The enzyme substrate, 2′,3′-cyclic adenosine monophosphate, had no effect on the oxidation of exogenous β-hydroxybutyrate or succinate by intact mitochondria. These findings suggest that 2′,3′-cyclic nucleotide 3′phosphohydrolase is more widely distributed than was previously thought and that the enzyme may play a fundamental role in membranes, independent of their specialized structure or functions.  相似文献   

14.
We have previously shown that 2,3-cyclic nucleotide 3-phosphodiesterase (CNP; EC 3.1.4.37) in rat central nervous tissues can be immunohistochemically stained with anti-bovine CNP serum. However, the anti-bovine CNP serum prepared in our laboratory has only weak cross-reactivity with rat CNP. Sections of bovine nervous tissues were found to be stained effectively with the serum, and the localization of CNP has been revealed in greater detail. We describe here the immunohistochemical localization of CNP in adult bovine cerebrum and cerebellum. CNP stained was localized in myelin sheaths, oligodendrocytes, and the processes of oligodendrocytes; astrocytes and neurons were negative. All myelinated nerve fibers appeared to be stained with the anti-CNP serum. Perineuronal and perivascular oligodendrocytes, and oligodendrocytes extending their processes to isolated myelin fibers were stained. Interfascicular oligodendrocytes, however, did not react or reacted faintly to the anti-CNP serum; only their processes were reactive. Comparison with the stain for S-100 protein was helpful to distinguish oligodendrocytes from astrocytes particularly when both glial cells were situated together at the perineuronal and perivascular positions.Dedicated to Professor Yasuzo Tsukada.  相似文献   

15.
In order to study the genealogical relationships among four groups (I to IV) of RNA coliphages, we sequenced 200 to 260 nucleotides from the 3′ termini of 14 phage RNAs according to the method of Sanger et al. (1977), and compared the results. It was found that the sequences of phage RNAs in the same group were extremely homologous (about 90%). On the other hand, when the sequences were compared with those from other groups, they were seen to be only about 50 to 60% homologous between group I and group II, and about 50% homologous between group III and group IV. In other combinations, such as groups I (or II) and III, and groups I (or II) and IV, however, the extent of homology was small. Furthermore, the sequences up to 30 residues from the 3′ end were found to be about 90% homologous between groups I and II, and between groups III and IV.These results confirm our previous findings, that the sequences located in the proximity of the 3′ end of phage RNA in the same group were well-conserved (Inokuchi et al., 1979), and that close relationships exist between groups I and II, and between groups III and IV (Furuse et al., 1979).  相似文献   

16.
17.
The relative levels of the central nervous system myelin marker enzyme 2:3-cyclic nucleotide 3-phosphodiesterase (EC 3.1.4.37, CNPase) were determined in neuroblastoma, astrocyte, oligodendrocyte and Schwann cell cultures and in freshly isolated human lymphocytes and platelets. The highest specific activities were associated with the cells that elaborate myelin membrane in the central and peripheral nervous system, oligodendrocytes and Schwann cells, respectively. Antiserum to bovine CNPase recognized both CNP1 and CNP2 in CNS myelin and human oligodendroglioma. In addition, a 53,000 dalton protein was evident on autoradiographs of immunoblotted PNS myelin and human oligodendroglioma proteins. Cultured rat oligodendrocyte, C6 and mouse NA neuroblastoma CNPase appear to share common determinants with the corresponding normal rat CNS enzyme.  相似文献   

18.
Abstract

A novel nucleoside analogue, 2′-naphthylmethyl-2′-deoxytubercidine, is synthesized and incorporated in oligonucleotides that stabilize bulges in partially complementary RNA.  相似文献   

19.
Abstract

The advent of AIDS has prompted the search for effective anti-HIV-1 agents, and, in view of the efficacy of azidothymidine in the treatment of AIDS, 2′,3′-dideoxynucleosides and analogues thereof have been considered as the most obvious candidates for AIDS chemotherapy. Various substituents have been introduced at the 3′-position, but only the 3′-azido and 3′-fluoro derivatives were found active against HIV-1. Introduction of a fluorine in organic compounds frequently causes a dramatic change in their biological activity. The stability of the carbon-fluorine bond and the strong electronegative character of fluorine, altering the electronic properties of the substituted molecule, led us to synthesize dideoxynucleosides with a fluorine substituent at different positions. The synthe-  相似文献   

20.
We investigated the actions of human recombinant α2-interferon and a secondary messenger of its action, 2′-5′-oligoadenylate, on tetrodotoxin (TTX)-sensitive sodium transport using human (IMR-32) and murine (NIE-115) neuroblastoma cells. In biochemical experiments using22Na, human interferon was shown to increase entry of22Na into IMR-32 neuroblastoma cells through the channels activated by veratrine and scorpion toxin. This increase was clearly dose-dependent. Cell treatment with TTX completely inhibited this sodium transport. On the contrary, 2′-5′-oligoadenylate depressed entry of22Na into neuroblastoma cells. The activation effect was not observed under the action of human α2-interferon on TTX-sensitive sodium flows to the murine neuroblastoma cells, which demonstrated the species-related specificity of this agent.  相似文献   

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