Crystal structure of the 2'-5' RNA ligase from Thermus thermophilus HB8

The 2'-5' RNA ligase family members are bacterial and archaeal RNA ligases that ligate 5' and 3' half-tRNA molecules with 2',3'-cyclic phosphate and 5'-hydroxyl termini, respectively, to the product containing the 2'-5' phosphodiester linkage. Here, the crystal structure of the 2'-5' RNA ligase protein from an extreme thermophile, Thermus thermophilus HB8, was solved at 2.5A resolution. The structure of the 2'-5' RNA ligase superimposes well on that of the Arabidopsis thaliana cyclic phosphodiesterase (CPDase), which hydrolyzes ADP-ribose 1",2"-cyclic phosphate (a product of the tRNA splicing reaction) to the monoester ADP-ribose 1"-phosphate. Although the sequence identity between the two proteins is remarkably low (9.3%), the 2'-5' RNA ligase and CPDase structures have two HX(T/S)X motifs in their corresponding positions. The HX(T/S)X motifs play important roles in the CPDase activity, and are conserved in both the CPDases and 2'-5' RNA ligases. Therefore, the catalytic mechanism of the 2'-5' RNA ligase may be similar to that of the CPDase. On the other hand, the electrostatic potential of the cavity of the 2'-5' RNA ligase is positive, but that of the CPDase is negative. Furthermore, in the CPDase, two loops with low B-factors cover the cavity. In contrast, in the 2'-5' RNA ligase, the corresponding loops form an open conformation and are flexible. These characteristics may be due to the differences in the substrates, tRNA and ADP-ribose 1",2"-cyclic phosphate.     

5'-modified agonist and antagonist (2'-5')(A)n analogues: synthesis and biological activity

Two 5'-modified (2'-5')(A)4 oligomers with an increased resistance to phosphatase degradation were synthesized and evaluated for their ability to develop an antiviral response when introduced into intact cells by microinjection or by chemical conjugation to poly(L-lysine). The enzymatic synthesis of 5'-gamma-phosphorothioate and beta,gamma-difluoromethylene (2'-5')(A)4 from adenosine 5'-O-(3-thiotriphosphate) and adenosine beta,gamma-difluoromethylenetriphosphate by (2'-5')-oligoadenylate synthetase is described. The isolation and characterization of these (2'-5')(A)4 analogues were achieved by high-performance liquid chromatography. The structures of 5'-modified tetramers were corroborated by enzyme digestion. These two 5'-modified tetramers compete as efficiently as natural (2'-5')(A)4 for the binding of a radiolabeled (2'-5')(A)4 probe to ribonuclease (RNase) L. Nevertheless, at the opposite to 5'-gamma-phosphorothioate (2'-5')(A)4, beta,gamma-difluoromethylene (2'-5')(A)4 failed to induce an antiviral response after microinjection in HeLa cells. In addition, it behaves as an antagonist of RNase L as demonstrated by its ability to inhibit the antiviral properties of 5'-gamma-phosphorothioate (2'-5')(A)4 when both are microinjected in HeLa cells. The increased metabolic stability of 5'-gamma-phosphorothioate (2'-5')(A)4 as compared to that of (2'-5')(A)4 was first demonstrated in cell-free extracts and then confirmed in intact cells after introduction in the form of a conjugate to poly(L-lysine). Indeed, 5'-gamma-phosphorothioate (2'-5')(A)4-poly(L-lysine) conjugate induces protein synthesis inhibition and characteristic ribosomal RNA cleavages for longer times than unmodified (2'-5')(A)4-poly(L-lysine) in the same cell system.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Expected distance between terminal nucleotides of RNA secondary structures

In "The ends of a large RNA molecule are necessarily close", Yoffe et al. (Nucleic Acids Res 39(1):292-299, 2011) used the programs RNAfold [resp. RNAsubopt] from Vienna RNA Package to calculate the distance between 5' and 3' ends of the minimum free energy secondary structure [resp. thermal equilibrium structures] of viral and random RNA sequences. Here, the 5'-3' distance is defined to be the length of the shortest path from 5' node to 3' node in the undirected graph, whose edge set consists of edges {i, i + 1} corresponding to covalent backbone bonds and of edges {i, j} corresponding to canonical base pairs. From repeated simulations and using a heuristic theoretical argument, Yoffe et al. conclude that the 5'-3' distance is less than a fixed constant, independent of RNA sequence length. In this paper, we provide a rigorous, mathematical framework to study the expected distance from 5' to 3' ends of an RNA sequence. We present recurrence relations that precisely define the expected distance from 5' to 3' ends of an RNA sequence, both for the Turner nearest neighbor energy model, as well as for a simple homopolymer model first defined by Stein and Waterman. We implement dynamic programming algorithms to compute (rather than approximate by repeated application of Vienna RNA Package) the expected distance between 5' and 3' ends of a given RNA sequence, with respect to the Turner energy model. Using methods of analytical combinatorics, that depend on complex analysis, we prove that the asymptotic expected 5'-3' distance of length n homopolymers is approximately equal to the constant 5.47211, while the asymptotic distance is 6.771096 if hairpins have a minimum of 3 unpaired bases and the probability that any two positions can form a base pair is 1/4. Finally, we analyze the 5'-3' distance for secondary structures from the STRAND database, and conclude that the 5'-3' distance is correlated with RNA sequence length.     

Purification of a RNA debranching activity from HeLa cells

The splicing of messenger RNA precursors (pre-mRNA) of eukaryotic cells involves the formation of a branched RNA intermediate known as a RNA lariat. This structure is formed in the first step of the reaction when a cleavage at the 5' splice site generates the 5' exon and a RNA species containing the intron and 3' exon in which the phosphate moiety at the 5' end of the intron is forming a 2'-5' phosphodiester bond with the 2'-hydroxyl moiety of a specific adenine residue near the 3' end of the intron forming a RNA branch with the following structure: -pA2'-pX-3'-pZ-. We have purified a debranching activity approximately 700-fold from the cytosolic fraction of HeLa cells. This activity catalyzes the hydrolysis of the 2'-5' phosphodiester bond of branched RNA structures yielding a 5'-phosphate end and a 2'-hydroxyl group at the branch attachment site. The activity possessed a sedimentation coefficient of 3.5 S. The reaction catalyzed by the purified fraction requires a divalent cation and is optimal at pH 7.0. The purified activity can efficiently hydrolyze triester trinucleotide structures (pY2'-pX-3'-pZ-) prepared by digestion of RNA lariats with nuclease P1. In contrast, a 2' phosphate monoester product (-pG2'-p 3'-pC-), formed by the wheat germ RNA ligase, was not attacked.     

Influence of internucleotide phosphate linkage on relative base stacking in 3'-5' and 2'-5' RNA: a circular dichroic spectroscopic study of RNA hexamer AACCUU.

The variations in base stacking interactions of two isomeric RNA hexamers, 3'-5'r (AACCUU) and 2'-5'r' (AACCUU), have been studied using temperature dependent CD spectroscopy. Both RNA hexamers, in single strand form, exhibited a right handed helical sense. Van't Hoff analysis of the CD spectral results, derived from a two state model, gave a higher enthalpy of stacking for 3'-5' RNA than for 2'-5'RNA. The results suggest that 3'-5' linkage in RNA facilitates formation of better helical stacks in relation to an isomeric 2'-5' linkage.     

Directing the outcome of deoxyribozyme selections to favor native 3'-5' RNA ligation

Previous experiments have identified numerous RNA ligase deoxyribozymes, each of which can synthesize either 2',5'-branched RNA, linear 2'-5'-linked RNA, or linear 3'-5'-linked RNA. These products may be formed by reaction of a 2'-hydroxyl or 3'-hydroxyl of one RNA substrate with the 5'-triphosphate of a second RNA substrate. Here the inherent propensities for nucleophilic reactivity of specific hydroxyl groups were assessed using RNA substrates related to the natural sequences of spliceosome substrates and group II introns. With the spliceosome substrates, nearly half of the selected deoxyribozymes mediate a ligation reaction involving the natural branch-point adenosine as the nucleophile. In contrast, mostly linear RNA is obtained with the group II intron substrates. Because the two sets of substrates differ at only three nucleotides, we conclude that the location of the newly created ligation junction in DNA-catalyzed branch formation depends sensitively on the RNA substrate sequences. During the experiment that led primarily to branched RNA, we abruptly altered the selection strategy to demand that the deoxyribozymes create linear 3'-5' linkages by introducing an additional selection step involving the 3'-5'-selective 8-17 deoxyribozyme. Although no 3'-5' linkages (相似文献   

Diprenylated chalcones and other constituents from the twigs of Dorstenia barteri var. subtriangularis

The twigs of Dorstenia barteri var. subtriangularis yielded three diprenylated chalcones: (-)-3-(3,3-dimethylallyl)-5'-(2-hydroxy-3-methylbut-3-enyl)-4,2',4'-trihydroxychalcone, (+)-3-(3,3-dimethylallyl)-4',5'-[2'-(1-hydroxy-1-methylethyl)-dihydrofurano]-4,2'-dihydroxychalcone and 3,4-(6",6"-dimethyldihydropyrano)-4',5'-[2',-(1-hydroxy-1-methylethyl)-dihydrofurano]-2'-hydroxychalcone for which the names bartericins A, B and C, respectively, are proposed. Stipulin, beta-sitosterol and its 3-beta-D-glucopyranosyl derivative were also isolated. The structures of these secondary metabolites were determined on the basis of spectroscopic analysis, especially, NMR spectra in conjunction with 2D experiments, COSY, HMQC and HMBC. The structural relationship of bartericins B and C was further established by the chemical cyclization of one to the other.     

Synthesis of modified RNA-oligonucleotides for structural investigations

RNA exhibits a higher structural diversity than DNA and is an important molecule in biology of life. It shows a number of secondary structures such as duplexes, hairpin loops, bulges, internal loops etc. However, in natural RNA, bases are limited to the four predominant structures U, C, A, and G and so the number of compounds that can be used for investigation of parameters of base stacking, base pairing and hydrogen bond, is limited. We synthesized different fluoromodifications of RNA building blocks: 1'-deoxy-1'-(2,4,6-trifluorophenyl)-beta-D-ribofuranose (F), 1'-deoxy-1'-(2,4,5-trifluorophenyl)-beta-D-ribofuranose (M) and 1'-deoxy-1'-(5-trifluoromethyl-1H-benzimidazol-1-yl)-beta-D-ribofuranose (D). Those amidites were incorporated and tested in a defined A, U-rich RNA sequence (12-mer, 5'-CUU UUC XUU CUU-3' paired with 3'-GAA AAG YAA GAA-5') (Schweitzer, B.A.; Kool, E.T. Aromatic nonpolar nucleosides as hydrophobic isosters of pyrimidine and purine nucleosides. J. Org. Chem. 1994, 59, 7238 pp.). Only one position was modified, marked as X and Y respectively. UV melting profiles of those oligonucleotides were measured.     

Increased stability and antiviral activity of 2'-O-phosphoglyceryl derivatives of (2'-5')oligo(adenylate)

Metabolically stable analogues of (2'-5')oligo(adenylate), (2'-5')(A)n, might constitute a new class of antiviral agents as they mimic some of the effects of interferons. 2'-O-phosphoglyceryl derivatives of (2'-5')(A)n oligomers, (2'-5')(A)n-PGro have been synthesized by chemical modification of their terminal ribose residue. Such analogues are resistant to degradation by phosphodiesterases but remain sensitive to phosphatase activity, at least in cell-free extracts. In line with its increased stability, (2'-5')(A)n-PGro has a powerful antiviral activity against an RNA virus when microinjected with micropipettes into the cytoplasm of intact cells. This antiviral activity remains transient however, possibly as a consequence of degradation in intact cells. Since (2'-5')(A)n and its derivatives do not easily cross cell membranes, their possible use in antiviral chemotherapy is tightly linked with the development of vectors suitable for their administration in vivo.     

NMR solution structures of [d(GCGAAT-3'-3'-alphaT-5'-5'-CGC)2] and its unmodified control.

We present the high-resolution solution structures of a self-complementary DNA decamer duplex featuring a single alpha-anomeric nucleotide per strand encompassed by a set of 3'-3' and 5'-5' phosphodiester linkages, d(GCGAAT-3'-3'-alphaT-5'-5'-CGC)2, alphaT, and its unmodified control, d(GCGAATTCGC)2, obtained by restrained molecular dynamics. Interproton distance and deoxyribose ring torsion angle restraints were deduced from homonuclear NOESY and DQF-COSY data, respectively. For both the control and alphaT duplexes, excellent global convergence was observed from two different (A- and B-) starting models. The final average structures of the two duplexes are highly homologous, and overall possess the traits characteristic of right-handed B-DNA duplexes. However, localized differences between the two structures stem from the enhanced conformational exchange in the deoxyribose ring of the cytidine following the 5'-5' linkage, the C3'- exo pseudorotation phase angle of the alpha-nucleotide, and unusual backbone torsions in the 3'-3' and 5'-5' phosphodiester linkages. The structural data reported here are relevant to the design of antisense therapeutics comprised of these modifications.     

Non-enzymatic, template-directed ligation of 2'-5' oligoribonucleotides. Joining of a template and a ligator strand.

Decauridylate containing exclusively a 2'-5' phospho-diester bond ([2'-5']U10) served as a template for the synthesis of oligoadenylates [oligo(A)s] from the 5'-phosphorimidazolide of 2'-5' diadenylate (ImpA-2'p5'A). Joining of [2'-5']U10and ImpA2'p5'A also took place in substantial amounts to yield long-chain oligoribonucleotides in the template-directed reaction. An unusual CD spectrum ascribed to helix formation between [2'-5']U10and [2'-5'](pA)2was observed under the same conditions as that of the template-directed reaction. The 3'-5' linked decauridylate ([3'-5']U10) also promoted the template-directed synthesis of oligo(A)s from ImpA2'p5'A, but more slowly compared with [2'-5']U10. The results indicate that short-chain RNA oligomers with a 2'-5' phosphodiester bond could lead to longer oligoribonucleotides by template-directed chain elongation.     

Interferon action: binding of viral RNA to the 40-kilodalton 2''-5''-oligoadenylate synthetase in interferon-treated HeLa cells infected with encephalomyocarditis virus.

The 40-kDa 2'-5'-oligoadenylate [(2'-5') (A)n] synthetase isoenzyme was proven to be a mediator of the inhibition of encephalomyocarditis virus (EMCV) replication by interferon (IFN). When activated by double-stranded RNA, this enzyme converts ATP into 2'-5'-oligoadenylate [(2'-5') (A)n], and (2'-5') (A)n was found to accumulate in IFN-treated, EMCV-infected cells. The only known function of (2'-5') (A)n is the activation of RNase L, a latent RNase, and this was also implicated in the inhibition of EMCV replication. Intermediates or side products in EMCV RNA replication, presumed to be partially double stranded, were shown to activate (2'-5') (A)n synthetase in vitro. These findings served as the basis of the long-standing hypothesis that the activator of (2'-5') (A)n synthetase in IFN-treated, EMCV-infected cells is the viral RNA. To test this hypothesis, we have generated a polyclonal rabbit antiserum to the human 40-kDa (2'-5') (A)n synthetase. The antiserum immunoprecipitated, from IFN-treated HeLa cells that had been infected with EMCV, the 40-kDa (2'-5') (A)n synthetase protein in complex with both strands of EMCV RNA. The immunoprecipitate was active in (2'-5') (A)n synthesis even without addition of double-stranded RNA, whereas the immunoprecipitate from IFN-treated, uninfected cells was not. These and other results demonstrate that in IFN-treated, EMCV-infected cells, viral RNA is bound to the (2'-5') (A)n synthetase and suggest that the agent activating the (2'-5') (A)n synthetase is the bound viral RNA.     

Association of 2''-5'' oligoribonucleotides.

Oligoribonucleotides with 2'-5' linkages have been synthesized on solid support. UV melting and CD experiments indicate complementary strands associate to give complexes with melting temperatures 30 to 40 degrees C lower than for duplexes formed by 3'-5' oligoribonucleotides with the same sequence. UV melting and imino proton NMR spectra and NOEs for (2'-5') CGGCGCCG are consistent with formation of an antiparallel duplex. The results suggest greater duplex stability was one factor favoring 3'-5' over 2'-5' linkages in evolution.     

5'-3' RNA-RNA interaction facilitates cap- and poly(A) tail-independent translation of tomato bushy stunt virus mrna: a potential common mechanism for tombusviridae

Tomato bushy stunt virus (TBSV) is the prototypical member of the genus Tombusvirus in the family Tombusviridae. The (+)-strand RNA genome of TBSV lacks both a 5' cap and a 3' poly(A) tail and instead contains a 3'-terminal RNA sequence that acts as a cap-independent translational enhancer (3' CITE). In this study, we have determined the RNA secondary structure of the translation-specific central segment of the 3' CITE, termed region 3.5 (R3.5). MFOLD structural modeling combined with solution structure mapping and comparative sequence analysis indicate that R3.5 adopts a branched structure that contains three major helices. Deletion and substitution studies revealed that two of these extended stem-loop (SL) structures are essential for 3' CITE activity in vivo. In particular, the terminal loop of one of these SLs, SL-B, was found to be critical for translation. Compensatory mutational analysis showed that SL-B functions by base pairing with another SL, SL3, in the 5' untranslated region of the TBSV genome. Thus, efficient translation of TBSV mRNA in vivo requires a 5'-3' RNA-RNA interaction that effectively circularizes the message. Similar types of interactions are also predicted to occur in TBSV subgenomic mRNAs between their 5' untranslated regions and the 3' CITE, and both genomic and subgenomic 5'-3' interactions are well conserved in all members of the genus Tombusvirus. In addition, a survey of other genera in Tombusviridae revealed the potential for similar 5'-3' RNA-RNA-based interactions in their viral mRNAs, suggesting that this mechanism extends throughout this large virus family.     

Structural, dynamic, and enzymatic properties of mixed alpha/beta-oligonucleotides containing polarity reversals

We employ NMR structure determination, thermodynamics, and enzymatics to uncover the structural, thermodynamic and enzymatic properties of alpha/beta-ODNs containing 3'-3' and 5'-5' linkages. RNase H studies show that alpha/beta-gapmers that are designed to target erbB-2 efficiently elicit RNase H activity. NMR structures of DNA.DNA and DNA.RNA duplexes reveal that single alpha-anomeric residues fit well into either duplex, but alter the dynamic properties of the backbone and deoxyriboses as well as the topology of the minor groove in the DNA.RNA hybrid.     

The effect of (2''-5'') and (3''-5'') phosphodiester linkages on conformational and stacking properties of cytidylyl-cytidine in aqueous solution.

Conformational properties of (2'-5') and (3'-5') CpC have been determined by proton magnetic resonance spectroscopy at 220 MHz. The ribose ring structures are predominantly 3E with the exception of the ring from the 2'-phosphate fragment of C(2'-5')pC which exhibits an 2E pucker. Bases are oriented anti with respect to the ribose and the conformations about C4'-C5', C5'-O5', C3'-O3' (C2'-O2') are gg, g'g', and g+ in equilibrium g-, respectively. The dimers exist as mixtures of stacked (g+g+ and g-g- about the P-O(C) bonds) and unstacked species at 20 degrees C. Stacking is estimated to be 35% in both dimers.