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At least 1,333 records · Page 74Linked to original sources
PubMed1991

Specific contacts between the bacteriophage T3, T7, and SP6 RNA polymerases and their promoters.

The specificity and structural simplicity of the bacteriophage T3, T7, and SP6 RNA polymerases make these enzymes particularly well suited for studies of polymerase-promoter interactions. To understand the initial recognition process between the enzyme and its promoters, DNA fragments that carry phage promoters were chemically modified by three different methods: base methylation, phosphate ethylation, and base removal. The positions at which these modifications prevented or enhanced binding by the RNA polymerases were then determined. The results indicate that specific contacts within the major groove of the promoter between positions-5 and -12 are important for phage polymerase binding. Removal of individual bases from either strand of the initiation region (-5 to +3) resulted in enhanced binding of the polymerase, suggesting that disruption of the helix in this region may play a role in stabilization of the polymerase-promoter complexes.

Base Sequence↗
PubMed1999

Prospective evaluation of amplification-boosted ELISA for heat-denatured p24 antigen for diagnosis and monitoring of pediatric human immunodeficiency virus type 1 infection.

The performance in pediatric human immunodeficiency virus type 1 (HIV-1) infection of a signal-amplification boosted ELISA for HIV-1 p24 antigen in plasma after heat-mediated immune complex dissociation was prospectively compared with polymerase chain reaction-based procedures. Diagnostic sensitivity and specificity of the p24 antigen test were 100% and 99.2%, respectively. Quantification revealed RNA in 85.7% and p24 antigen in 87.4% of 230 samples from 25 infected children. Concentrations of these indices in individual samples correlated (P<.0001). Introduction or modification of antiretroviral treatment showed concordant responses of RNA and p24 antigen in 39 (90.7%) of 43 instances. The treatment-induced changes in concentrations of RNA were higher than those of p24 antigen in 11 instances. In 1 instance, however, the concentration change of p24 antigen was greater than that of RNA (P=. 002). Variation of RNA concentrations was more marked than that of p24 antigen (P=.002). The p24 antigen test was equivalent to PCR for diagnosing and monitoring pediatric HIV-1 infection.

Anti-HIV Agents↗
PubMed2006

Palmitoylation and polymerization of hepatitis C virus NS4B protein.

Hepatitis C Virus (HCV) NS4B protein induces a specialized membrane structure which may serve as the replication platform for HCV RNA replication. In the present study, we demonstrated that NS4B has lipid modifications (palmitoylation) on two cysteine residues (cysteines 257 and 261) at the C-terminal end. Site-specific mutagenesis of these cysteine residues on individual NS4B proteins and on an HCV subgenomic replicon showed that the lipid modifications, particularly of Cys261, are important for protein-protein interaction in the formation of the HCV RNA replication complex. We further demonstrated that NS4B can undergo polymerization. The main polymerization determinants were mapped in the N-terminal cytosolic domain of NS4B protein; however, the lipid modifications on the C terminus also facilitate the polymerization process. The lipid modification and the polymerization activity could be two properties of NS4B important for its induction of the specialized membrane structure involved in viral RNA replication.

Cysteine↗
PubMed1975

Modification of the response to 8 MeV electrons of auxotrophic strains of Escherichia coli by amino-acid deprivation and rifampicin.

An investigation has been made of the possibility that the accumulation of RNA enriched in messenger can influence the survival of bacteria after exposure to 8 MeV electrons. The multiple amino-acid auxotroph E. coli CP79 RCrel accumulates RNA during inhibition of protein synthesis, and the proportion of RNA present as messenger increases by a factor of 3 during a 2-hour period of amino-acid deprivation. Amino-acid withdrawal led to changes in the shape of the survival curve, which also occurred in strain CP78 RCstr, which synthesizes very little RNA when deprived of amino acids. We conclude that accumulation of RNA enriched in messenger plays little or no part in modification of the survival of irradiated bacteria. We show that rifampicin, an inhibitor of RNA synthesis, increases the sensitivity of both strains equally.

Amino Acids↗
PubMed2006

Single-stranded small interfering RNA are more immunostimulatory than their double-stranded counterparts: a central role for 2'-hydroxyl uridines in immune responses.

It has recently become apparent that certain small interfering RNA (siRNA) sequences stimulate the innate immunity through endosomal Toll-like receptors (TLR), particularly TLR7 and TLR8. However, it remains unclear whether siRNA duplexes act as specific ligands for these receptors. To address this question and to overcome the problem of immune activation by siRNA, several RNA sequences were chemically synthesized and their effects were investigated. Results indicate that human peripheral blood mononuclear cells (PBMC) recognize and respond to a large number of sense or antisense single-stranded (ss) siRNA. In most cases immunostimulatory RNA motifs are more effectively recognized by innate immunity in the context of ss siRNA as compared to siRNA duplexes. Novel immunostimulatory RNA motifs were identified and their replacement with adenosines abrogated immune activation. Most notably, replacement of the 2'-hydroxyl uridines with either 2'-fluoro, 2'-deoxy or 2'-O-methyl uridines abrogated immune activation. Thus, immune recognition of RNA by TLR can be evaded by 2'-ribose modifications of only uridines. Collectively, the data should facilitate the development of siRNA therapeutics and expand the understanding of how RNA is sensed by innate immunity.

Adjuvants, Immunologic↗
PubMed1984

Effect of S-adenosylmethionine on human rotavirus RNA synthesis.

The characteristics of human rotavirus-associated RNA polymerase activity have been examined in relation to the effects of ribonucleoside triphosphate analogs and S-adenosylmethionine. These effects were analyzed by testing two forms of activated virus particles: EDTA- and heat-treated virions. The former lack outer shell proteins, and activation by means of heat treatment does not introduce any apparent modification in virion structure. Virus-associated RNA polymerase shows similar properties in both preparations, suggesting that outer proteins are not directly involved in RNA synthesis. Transcription in this virus is specifically dependent on a hydrolyzable form of ATP. Such a requirement is not overcome by preincubation or by the addition of S-adenosylmethionine, suggesting a hypothetical mechanism that couples transcription to ATP hydrolysis. The addition of S-adenosylmethionine stimulated transcription and diminished the Km value not only for ATP but also for the other three ribonucleoside triphosphates. Analysis of methylated RNA products suggested that methyl groups were incorporated into all of the RNA species synthesized by virion-associated polymerase. Further analysis of those RNA molecules showed that they contained cap structures at the 5' end. The results suggest that the cap structure at the end of RNA molecules may enable RNA polymerase to elongate transcripts more efficiently, in a reaction in which the hydrolysis of ATP is involved.

Feces↗
PubMed1998

Identification of two Escherichia coli pseudouridine synthases that show multisite specificity for 23S RNA.

Several putative Escherichia coli pseudouridine (Psi) synthases have been identified by iterative searching of genomic databases for ORFs homologous to known Psi synthases [Gustafsson et al. (1996) Nucleic Acids Res. 24, 3756-3762]. Of these, yceC and yfiI were proposed to encode Psi synthases which modify 23S rRNA. In the present work, yceC and yfiI were cloned and overexpressed in E. coli, and the encoded enzymes, YceC and YfiI, were purified to homogeneity. Both proteins converted Urd residues of rRNA to Psi, thus confirming their identities as Psi synthases. However, in in vitro experiments both enzymes extensively modified Urd residues of both 23S rRNA and 16S rRNA. Gene-disruption of yceCresulted in the absence of Psi modification at positions U955, 2504, and 2580 of 23S RNA, thus identifying these sites as in vivo targets for YceC. Likewise, yfiI disruption resulted in the absence of Psi modification at positions U1911, 1917, and possibly 1915 of 23S RNA. Disruption of yceC did not affect the growth under the conditions tested, whereas yfiI-disrupted cells showed a dramatic decrease in growth rate. Since YceC and YfiI hypermodify RNA in vitro, factors in addition to ribonucleotide sequence must contribute to the in vivo specificity of these enzymes.

Bacterial Proteins↗
PubMed2003

Influence of temperature on tRNA modification in archaea: Methanococcoides burtonii (optimum growth temperature [Topt], 23 degrees C) and Stetteria hydrogenophila (Topt, 95 degrees C).

We report the first study of tRNA modification in psychrotolerant archaea, specifically in the archaeon Methanococcoides burtonii grown at 4 and 23 degrees C. For comparison, unfractionated tRNA from the archaeal hyperthermophile Stetteria hydrogenophila cultured at 93 degrees C was examined. Analysis of modified nucleosides using liquid chromatography-electrospray ionization mass spectrometry revealed striking differences in levels and identities of tRNA modifications between the two organisms. Although the modification levels in M. burtonii tRNA are the lowest in any organism of which we are aware, it contains more than one residue per tRNA molecule of dihydrouridine, a molecule associated with maintenance of polynucleotide flexibility at low temperatures. No differences in either identities or levels of modifications, including dihydrouridine, as a function of culture temperature were observed, in contrast to selected tRNA modifications previously reported for archaeal hyperthermophiles. By contrast, S. hydrogenophila tRNA was found to contain a remarkable structural diversity of 31 modified nucleosides, including nine methylated guanosines, with eight different nucleoside species methylated at O-2' of ribose, known to be an effective stabilizing motif in RNA. These results show that some aspects of tRNA modification in archaea are strongly associated with environmental temperature and support the thesis that posttranscriptional modification is a universal natural mechanism for control of RNA molecular structure that operates across a wide temperature range in archaea as well as bacteria.

Cell Division↗
PubMed2002

alpha-L-ribo-configured locked nucleic acid (alpha-L-LNA): synthesis and properties.

The syntheses of monomeric nucleosides and 3'-O-phosphoramidite building blocks en route to alpha-L-ribo-configured locked nucleic acids (alpha-L-LNA), composed entirely of alpha-L-LNA monomers (alpha-L-ribo configuration) or of a mixture of alpha-L-LNA and DNA monomers (beta-D-ribo configuration), are described and the alpha-L-LNA oligomers are studied. Bicyclic 5-methylcytosin-1-yl and adenine-9-yl nucleoside derivatives have been prepared and the phosphoramidite approach has been used for the automated oligomerization leading to alpha-L-LNA oligomers. Binding studies revealed very efficient recognition of single-stranded DNA and RNA target oligonucleotide strands. Thus, stereoirregular alpha-L-LNA 11-mers containing a mixture of alpha-L-LNA monomers and DNA monomers ("mix-mer alpha-L-LNA") were shown to display DeltaT(m) values of +1 to +3 degrees C per modification toward DNA and +4 to +5 degrees C toward RNA when compared with the corresponding unmodified DNA x DNA and DNA x RNA reference duplexes. The corresponding DeltaT(m) values per modification for the stereoregular fully modified alpha-L-LNA were determined to be +4 degrees C (against DNA) and +5 degrees C (against RNA). 11-Mer alpha-L-LNAs (mix-mer alpha- L-LNA or fully modified alpha- L-LNA) were shown in vitro to be significantly stabilized toward 3'-exonucleolytic degradation. A duplex formed between RNA and either mix-mer alpha-L-LNA or fully modified alpha-L-LNA induced in vitro Escherichia coli RNase H-mediated cleavage, albeit very slow, of the RNA targets at high enzyme concentrations.

Circular Dichroism↗
PubMed1984

[Characterization of total RNA, messenger poly(A)+ RNA and homologous complementary DNA in experimental cardiac hypertrophy].

Two biological modifications are observed during adaptation of cardiac tissue to work overload: an increase in total protein synthesis and a redistribution of myosin isoenzymes. These modifications suggest that changes in DNA transcription are involved in the early response of cardiac tissue to overload. Results are reported in this paper that show a parallel increase in total and polyadenylated RNA content and concentration in cardiac overloaded tissue in the rat. The characterization of cardiac poly (A)+ mRNA by mRNA X cDNA hybridization and the identification of specific mRNAs with recombinant plasmid cDNAs are given in this report as preliminary results.

Animals↗
PubMed2005

Fully grown mouse oocyte contains transcription inhibiting activity which acts through histone deacetylation.

Mouse ovarian oocytes remain transcriptionally active during the entire period of oocyte growth. In the fully grown antral oocytes, RNA synthesis is terminated before initiation of maturation. The mechanism involved in this process remains unknown. It was proposed that some signals provided by companion granulosa cells, and accumulating in the cytoplasm of the oocyte, play a role in the termination of RNA synthesis. It seems possible that under influence of these signals, the oocyte cytoplasm becomes transcriptionally nonpermissive. In the present study, transcriptionally active, single blastomeres of the 8-cell mouse embryo were fused with nontranscribing fully grown oocytes, and RNA synthesis was analyzed in the resulting hybrids. We show that after 27 hr of culture of hybrid cells, transcription in the blastomere nuclei was finally silenced. This process occurs without nuclear envelope breakdown and is accompanied by progressive condensation of the chromatin in the blastomere nuclei. We show also the involvement of core histone deacetylation in the modification of chromatin configuration and termination of RNA synthesis in the blastomere nuclei.

Animals↗
PubMed2006

Leaving a mark: the many footprints of the elongating RNA polymerase II.

The elongation phase of transcription by RNA polymerase II involves a complex choreography of events besides the polymerization of RNA. In addition to coordinating the processing of the nascent transcript, elongating RNA polymerase II recruits histone methyltransferases to methylate lysines 4 and 36 of histone H3 in nucleosomes in the body of actively transcribed genes. Methylation at these sites is genetically implicated in marking actively transcribed genes. Recent studies link transcriptional elongation by RNA polymerase II to H3K9 methylation and the recruitment of the HP1 family protein HP1gamma. These findings expand the role for RNA polymerase II elongation in targeting chromatin modifications to include a histone methyl mark more commonly associated with gene silencing.

Animals↗
PubMed1996

Oligodeoxynucleoside phosphoramidates (P-NH2): synthesis and thermal stability of duplexes with DNA and RNA targets.

Syntheses of non ionic oligodeoxynucleoside phosphoramidates (P-NH2) and mixed phosphoramidate- phosphodiester oligomers were accomplished on automated solid supported DNA synthesizer using both H-phosphonate and phosphoramidite chemistries, in combination with t-butylphenoxyacetyl for N-protection of nucleoside bases, an oxalyl anchored solid support and a final treatment with methanolic ammonia. Thermal stabilities of the hybrids formed between these new analogues and their DNA and RNA complementary strands were determined and compared with those of the corresponding unmodified oligonucleotides, as well as of the phosphorothioate and methylphosphonate derivatives. Dodecathymidines containing P-NH2 links form less stable duplexes with DNA targets, d(C2A12C2) (deltaTm/modification -1.4 degrees C) and poly dA (deltaTm/modification -1.1 degrees C) than the corresponding phosphodiester and methylphosphonate analogues, but the hybrids are slightly more stable than the one obtained with phosphorothioate derivative. The destabilization is more pronounced with poly rA as the target (deltaTm/modification -3 degrees C) and could be compared with that found with the dodecathymidine methylphosphonate. The modification is less destabilizing in an heteropolymer-RNA duplex (deltaTm/modification -2 degrees C). As expected, the P-NH2 modifications are highly resistant towards the action of various nucleases. It is also demonstrated that an all P-NH2 oligothymidine does not elicit Escherichia coli RNase H hydrolysis of the poly rA target but that the modification may be exploited in chimeric oligonucleotides combining P-NH2 sections with a central phosphodiester section.

Amides↗
PubMed2006

Electrospray ionization tandem mass spectrometry of biphenyl-modified oligo(deoxy)ribonucleotides.

Antisense oligonucleotides and aptamers are important candidates for future therapeutic applications. Different structural modifications are introduced into oligonucleotides to obtain high affinity and binding specificity. Sequence elucidation of oligonucleotides incorporating a wide variety of modifications presents an analytical challenge, as the standard protocols cannot be applied. Mass spectrometry has the potential to solve complex structural problems. However, a better understanding of the fundamental aspects of gas-phase dissociation of modified DNA and RNA is needed. In this work the influence of specific chemical modifications on backbone dissociation is pointed out. Biphenyl-modified oligo(deoxy)ribonucleotides, which incorporate C-glycosidic bound abasic nucleobase substitutes, were subjected to collision-induced dissociation in an electrospray tandem mass spectrometer, with the goal to investigate the role of nucleobase loss on backbone dissociation. DNA bearing biphenyl nucleobase substitutes show abundant [a-B]- and w-ions generated by cleavage of the 3'-C-O bonds, except for the phosphodiester groups adjacent to the biphenyl modifications. At these positions no dissociation was observed, demonstrating the dependence of DNA backbone dissociation on nucleobase loss. Also, no evidence for a base loss independent mechanism responsible for formation of w-ions was found. RNA incorporating biphenyl nucleobase substitutes fragment into c- and y-ions resulting from cleavage of the 5'-P-O bond. Adjacent to the biphenyl modifications no altered dissociation behavior was found. This leads to the conclusion that dissociation of RNA is independent of the 1'-modification and, therefore, independent of nucleobase loss.

Biphenyl Compounds↗
PubMed2005

N6-Acetyladenosine: a new modified nucleoside from Methanopyrus kandleri tRNA.

Post-transcriptionally modified nucleosides are constituents of transfer RNA (tRNA) that are known to influence tertiary structure, stability and coding properties. Modifications in unfractionated tRNA from the phylogenetically unique archaeal methanogen Methanopyrus kandleri (optimal growth temperature 98 degrees C) were studied using liquid chromatography-mass spectrometry to establish the extent to which they might differ from those of other methanogens. The exceptionally diverse population of nucleosides included four new nucleosides of unknown structure, and one that was characterized as N(6)-acetyladenosine, a new RNA constituent. The nucleoside modification pattern in M. kandleri tRNA is notably different from that of other archaeal methanogens, and is closer to that of the thermophilic crenarchaeota.

Adenosine↗
PubMed1995

An assessment of the antisense properties of RNase H-competent and steric-blocking oligomers.

The antisense activity and gene specificity of two classes of oligonucleotides (ONs) were directly compared in a highly controlled assay. One class of ONs has been proposed to act by targeting the degradation of specific RNAs through an RNase H-mediated mechanism and consists of C-5 propynyl pyrimidine phosphorothioate ONs (propyne-S-ON). The second class of antisense agents has been proposed to function by sterically blocking target RNA formation, transport or translation and includes sugar modified (2'-O-allyl) ONs and peptide nucleic acids (PNAs). Using a CV-1 cell based microinjection assay, we targeted antisense agents representing both classes to various cloned sequences localized within the SV40 large T antigen RNA. We determined the propyne-S-ON was the most potent and gene-specific agent of the two classes which likely reflected its ability to allow RNase H cleavage of its target. The PNA oligomer inhibited T Ag expression via an antisense mechanism, but was less effective than the propyne-S-ON; the lack of potency may have been due in part to the PNAs slow kinetics of RNA association. Interestingly, unlike the 2'-O-allyl ON, the antisense activity of the PNA was not restricted to the 5' untranslated region of the T Ag RNA. Based on these findings we conclude that PNAs could be effective antisense agents with additional chemical modification that will lead to more rapid association with their RNA target.

Allosteric Site↗
PubMed2002

Developments in RNA chemistry, a personal view.

This review describes some of the contributions of chemistry to the RNA field with a personal bias towards the phosphorothioate modification and the derivatives at the ribose 2'-position. The usefulness of these modifications is discussed and documented with some examples.

Animals↗