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PubMed1976

The binding site of protein L1 ON 23-S ribosomal RNA of Escherichia coli. 2. Identification of the rna region contained in the L1 ribonucleoproteins and determination of the order of the RNA subfragments within this region.

Ribonucleoproteins were obtained by T1 ribonuclease digestion of reconstitued complexes of ribosomal protein L1 AND 23-S RNA from Escherichia coli. The RNA region of the main ribonucleoprotein 2 was totally digested with T1 ribonuclease. The oligonucleotide products were characterised and they showed that this region comprises 148 nucleotides located between the 550th and 1000th necleotides from the 3' end of the 23-S RNA. Of the other two ribonucleoproteins, the largest ribonucleoprotein 1 contained an extra RNA sequence, of at least 15 nucleotides, that was located at the 5' end of the RNA region. The smallest ribonucleoprotein 3 lacked an RNA section towards the 3' end of the region. The order of the RNA subfragments and the enzymic cutting positions in the whole RNA region are given for the ribonucleoproteins. It is shown that protein L1 most strongly protects a continuous section of 115 nucleotides at the 5' end of the main RNA region. Finally, evidence is presented for a methylated base, and for two sequence heterogeneities, in this region of the 23-S RNA.

Alkaline Phosphatase↗
PubMed1989

Tentative identification of RNA-dependent RNA polymerases of dsRNA viruses and their relationship to positive strand RNA viral polymerases.

Amino acid sequence stretches similar to the four most conserved segments of positive strand RNA viral RNA-dependent RNA polymerases have been identified in proteins of four dsRNA viruses belonging to three families, i.e. P2 protein of bacteriophage phi 6 (Cystoviridae), RNA 2 product of infectious bursa disease virus (Birnaviridae), lambda 3 protein of reovirus, and VP1 of bluetongue virus (Reoviridae). High statistical significance of the observed similarity was demonstrated, allowing identification of these proteins as likely candidates for RNA-dependent RNA polymerases. Based on these observations, and on the previously reported sequence similarity between the RNA polymerases of a yeast dsRNA virus and those of positive strand RNA viruses, a possible evolutionary relationship between the two virus classes is discussed.

Amino Acid Sequence↗
PubMed2000

De novo RNA synthesis catalyzed by HCV RNA-dependent RNA polymerase.

The 65 kDa RNA-dependent RNA polymerase (NS5B), encoded by the hepatitis C virus (HCV) genome, is a key component involved in viral replication. Here we provide the direct evidence that purified HCV polymerase catalyzed de novo RNA synthesis in a primer-independent manner using homopolymers and HCV RNA as templates. The enzyme could utilize both polyC and polyU as templates for de novo RNA synthesis, suggesting that NS5B specifically recognized pyrimidine bases for initiation. More importantly, NS5B also catalyzed de novo RNA synthesis with an HCV RNA template; the resulting nascent RNA products, smaller than the template used, contained ATP as the first nucleotide. These results indicate that the newly synthesized RNAs did not result from template self-priming and suggest that a replication initiation site in the HCV RNA genome is a uridylate.

3' Untranslated Regions↗
PubMed2004

Viral RNA-directed RNA polymerases use diverse mechanisms to promote recombination between RNA molecules.

An earlier developed purified cell-free system was used to explore the potential of two RNA-directed RNA polymerases (RdRps), Qbeta phage replicase and the poliovirus 3Dpol protein, to promote RNA recombination through a primer extension mechanism. The substrates of recombination were fragments of complementary strands of a Qbeta phage-derived RNA, such that if aligned at complementary 3'-termini and extended using one another as a template, they would produce replicable molecules detectable as RNA colonies grown in a Qbeta replicase-containing agarose. The results show that while 3Dpol efficiently extends the aligned fragments to produce the expected homologous recombinant sequences, only nonhomologous recombinants are generated by Qbeta replicase at a much lower yield and through a mechanism not involving the extension of RNA primers. It follows that the mechanisms of RNA recombination by poliovirus and Qbeta RdRps are quite different. The data favor an RNA transesterification reaction catalyzed by a conformation acquired by Qbeta replicase during RNA synthesis and provide a likely explanation for the very low frequency of homologous recombination in Qbeta phage.

Allolevivirus↗
PubMed1999

Spatial perturbations within an RNA promoter specifically recognized by a viral RNA-dependent RNA polymerase (RdRp) reveal that RdRp can adjust its promoter binding sites.

RNA synthesis during viral replication requires specific recognition of RNA promoters by the viral RNA-dependent RNA polymerase (RdRp). Four nucleotides (-17, -14, -13, and -11) within the brome mosaic virus (BMV) subgenomic core promoter are required for RNA synthesis by the BMV RdRp (R. W. Siegel et al., Proc. Natl. Acad. Sci. USA 94:11238-11243, 1997). The spatial requirements for these four nucleotides and the initiation (+1) cytidylate were examined in RNAs containing nucleotide insertions and deletions within the BMV subgenomic core promoter. Spatial perturbations between nucleotides -17 and -11 resulted in decreased RNA synthesis in vitro. However, synthesis was still dependent on the key nucleotides identified in the wild-type core promoter and the initiation cytidylate. In contrast, changes between nucleotides -11 and +1 had a less severe effect on RNA synthesis but resulted in RNA products initiated at alternative locations in addition to the +1 cytidylate. The results suggest a degree of flexibility in the recognition of the subgenomic promoter by the BMV RdRp and are compared with functional regions in other DNA and RNA promoters.

Promoter Regions, Genetic↗
PubMedDate not supplied

[A new class of small RNP (alpha-RNP) containing antisense RNA in K-562 cells. II. The interaction of the RNA--the alpha-RNP component--with heterogeneous nuclear and messenger RNA in the normal state and under DMSO exposure].

Small antisense RNA (alpha-RNA), components of a new class of small nuclear and cytoplasmic RNP (alpha-RNP) identified in the cells of K-562 human proerythroleukemia cell line, are capable of hybridizing under stringent conditions with precursors of mRNA (heterogeneous nuclear RNA or mRNA) and with mRNA of these cells. We found that DMSO, an agent inducing differentiation in K-562 cells, is capable of regulating the composition of alpha-RNA population and concomitantly changes the content of mRNA that has regions homologous (complementary) to alpha-RNA. Specifically, it has been demonstrated that DMSO decreases the level of alpha-RNA, which hybridizes with the actin gene. Results of restriction mapping of regions of complementary interaction of alpha-RNA with the actin gene point out that alpha-RNA hybridizes with regions containing the promotor area and 3'-nontranslated area of the gene. It is proposed that small antisense alpha-RNA (alpha-RNP) participates in the control of gene expression at posttranscriptional level in cell cytoplasm.

Cell Differentiation↗
PubMed1997

Minimal templates directing accurate initiation of subgenomic RNA synthesis in vitro by the brome mosaic virus RNA-dependent RNA polymerase.

Short regions of (-)-strand brome mosaic virus (BMV) RNA3, proscripts, were shown to direct accurate in vitro synthesis of (+)-strand subgenomic RNA by the BMV RNA-dependent RNA polymerase (RdRp), facilitating characterization of the sequences and/or structures directing subgenomic RNA synthesis. Proscripts retaining fewer than 8-nt of an 18-nt polyuridylate tract located just upstream of the core promoter sequence (French R, Ahlquist P, 1988, J Virol 62:2411-2420; Marsh LE, Dreher TW, Hall TC, 1988, Nucleic Acids Res 16:981-995) directed dramatically less synthesis of 26-nt or longer products. Original levels of RNA synthesis were not restored by replacement of the 3' polyuridylate tract with polyadenylate, polycytidylate, or polyguanylate tracts, or by movement of the polyuridylate tract to the 5' end of the proscript. The polyuridylate tract presumably binds some component(s) required for RdRp activity because the addition of poly(U) [but not poly(C)] RNA to RdRp reactions decreased RNA synthesis significantly. Quite surprisingly, deletions of the polyuridylate tract in proscripts directing synthesis of 24-nt or shorter products had little or no detrimental effect on subgenomic RNA synthesis, correlated with their inability to form a computer-predicted stem-loop present in longer proscripts requiring the polyuridylate tract. Successive 3' and 5' deletions demonstrated that the minimal elements required for accurate initiation of subgenomic RNA synthesis are within a proscript of 22-nt.

Bromovirus↗
PubMed2001

RNA-catalyzed RNA polymerization: accurate and general RNA-templated primer extension.

The RNA world hypothesis regarding the early evolution of life relies on the premise that some RNA sequences can catalyze RNA replication. In support of this conjecture, we describe here an RNA molecule that catalyzes the type of polymerization needed for RNA replication. The ribozyme uses nucleoside triphosphates and the coding information of an RNA template to extend an RNA primer by the successive addition of up to 14 nucleotides-more than a complete turn of an RNA helix. Its polymerization activity is general in terms of the sequence and the length of the primer and template RNAs, provided that the 3' terminus of the primer pairs with the template. Its polymerization is also quite accurate: when primers extended by 11 nucleotides were cloned and sequenced, 1088 of 1100 sequenced nucleotides matched the template.

Base Sequence↗
PubMed1993

RNA duplex unwinding activity of poliovirus RNA-dependent RNA polymerase 3Dpol.

The ability of highly purified preparations of poliovirus RNA-dependent RNA polymerase, 3Dpol, to unwind RNA duplex structures was examined during a chain elongation reaction in vitro. Using an antisense RNA prehybridized to an RNA template, we show that poliovirus polymerase can elongate through a highly stable RNA duplex of over 1,000 bp. Radiolabeled antisense RNA was displaced from the template during the reaction, and product RNAs which were equal in length to the template strand were synthesized. Unwinding did not occur in the absence of chain elongation and did not require hydrolysis of the gamma-phosphate of ATP. The rate of elongation through the duplex region was comparable to the rate of elongation on the single-stranded region of the template. Parallel experiments conducted with avian myeloblastosis virus reverse transcriptase showed that this enzyme was not able to unwind the RNA duplex, suggesting that strand displacement by poliovirus 3Dpol is not a property shared by all polymerases.

Adenosine Triphosphate↗
PubMed1977

RNA methylation and control of eukaryotic RNA biosynthesis. Effects of cycloleucine, a specific inhibitor of methylation, on ribosomal RNA maturation.

The role of RNA methylation in the control of ribosome production in mammalian cells has been reinvestigated through a study of the effects in vivo of cycloleucine, a specific and reversible inhibitor of nucleic acid methylations. No close coupling is observed between methylation and transcription. During an extensive blocking of methylation, the synthesis of preribosomal 45-S RNA continues, although at a slightly reduced rate. Transcription and methylation can be temporarily uncoupled in vivo without impairing significantly the efficiency of the subsequent maturation of the transcript which takes place when the methylation is resumed. At the post-transcriptional level, two main observations are made. First, the cleavage pattern of ribosomal RNA is not qualitatively modified by the drug treatment. Preribosomal 45-S RNA which is synthesized during an extensive blocking of methylations (95%) is cleaved in vivo in a stepwise fashion into molecules resembling the species of the normal processing. Undermethylation does not result in the appearance of new size products and no extensive or rapid degredation of rRNA precursors can be detected. Second, the global efficiency of the process of ribosomal RNA maturation is severely affected, in quantitative terms; the undermethylation partially inhibits several stages in the maturation pathway and does not block selectively at a definite step of processing, as previously reported by others. Two main modifications are observed. (a) In the nucleus, the life times of the various undermethylated intermediates of ribosomal RNA maturation are markedly increased and a significant accumulation of these forms is detected by long-term labelling studies. (b) The rate of appearance of ribosomal 28-S RNA into the cytoplasm is severely affected (85-90% inhibition), in much higher proportions than the formation of its immediate precursor, nuclear 28-S RNA, thus suggesting a particular sensitivity to a lack of methylation of the last stage of preribosome maturation (possibly the release to cytoplasm). Finally, if a normal level of methylation does not appear to be stringently required for the completion of ribosomal RNA maturation, our results indicate that the degree of RNA methylation can modulate the general efficiency of the maturation processess.

Amino Acids↗
PubMed1987

Structure and accessibility of domain I of Escherichia coli 23 S RNA in free RNA, in the L24-RNA complex and in 50 S subunits. Implications for ribosomal assembly.

Domain I of 23 S RNA of Escherichia coli was probed in renatured RNA, in the protein L24-RNA complex and in 50 S subunits with ribonucleases specific for single- and double-stranded regions and with chemical reagents specific for guanosines (N-1 and N-2), adenosines (N-1, N-7 and N-6), cytidines (N-3) and uridines (N-3). Reactive sites were detected by a reverse transcriptase procedure. The results support most new features of the latest version of the Santa Cruz/Urbana model of the secondary structure, which is based on evidence from sequence comparison. Most double-helical segments were reactive to cobra venom ribonuclease to some degree; the exceptions were the five "long-range" helices that are probably compactly folded within the structure. The data provide evidence for the occurrence of A(syn) X G(anti) pairings in internal loops and at the ends of some helices; they also support the existence of extensive higher-order structuring, especially within the interhelical regions, and are compatible with two of three tertiary interactions in the free RNA that were predicted from comparative sequence studies. Protein L24 is the only primary binding protein that associates with domain I and it strongly protects two sites against ribonuclease and chemical activity. Site A has the properties of a classic protein binding site and we conclude from four lines of evidence that it is the primary attachment site. Site B is rich in highly conserved, unpaired adenosine residues and lies in a potentially critical region of the structure adjoining a group of long-range helices; we infer that L24 binding here is related to the important role of L24 in initiating ribosomal assembly; the existence of both sites is supported, independently, by genetic experiments. L24-induced enhanced reactivities were detected throughout the domain and are consistent with a general "tuning" of the RNA structure. The RNA domain in the 50 S subunits is almost completely resistant to ribonucleases and only a few sites, mainly interhelical, are accessible to chemical reagents. The appearance of several newly reactive nucleotides in the subunit RNA and the enhancement of some others suggest that some minor conformational changes occur on assembly. Nevertheless, the minimal secondary structure of the renatured RNA appears to be retained. We draw the general conclusion that domain I is a highly structured domain that is important for initiating assembly and for the subsequent organization of the ribosome.

Binding Sites↗
PubMed2006

Inhibition of hepatitis C virus RNA replication by short hairpin RNA synthesized by T7 RNA polymerase in hepatitis C virus subgenomic replicons.

RNA interference (RNAi) is a cellular process that induces gene silencing by which small duplexes of RNA specifically target a homologous sequence for cleavage by cellular ribonucleases. Here, to test the RNAi method for blocking hepatitis C virus (HCV) RNA replication, we created four short hairpin RNAs (shRNAs) targeting the HCV internal ribosome entry site/Core gene transcript using T7 RNA polymerase. shRNA suppressed the replication of HCV RNA in the HCV replicon. On the other hand, short interfering RNAs synthesized using the T7 RNA polymerase system trigger a potent induction of interferon-alpha and -beta in a variety of cells. We examined whether the shRNAs synthesized using the T7 RNA polymerase system activated double-stranded RNA-dependent protein kinase, 2'-5' oligoadenylate synthetase, or interferon-regulatory factor-3. Our results demonstrated that the T7-transcribed shRNA did not activate these proteins in Huh-7 cells and the HCV replicon. These shRNAs are a promising new strategy for anti-HCV gene therapeutics.

Base Sequence↗
PubMed1998

Site size of cooperative single-stranded RNA binding by poliovirus RNA-dependent RNA polymerase.

The poliovirus RNA-dependent RNA polymerase binds cooperatively to single-stranded RNA. We have determined the minimal RNA-binding site size of the poliovirus polymerase using binding titration with oligonucleotides of increasing length. A dramatic increase in affinity was observed when the length of the oligo(U) increased from 8 to 10 nucleotides (nt), arguing that the minimal size of RNA for polymerase binding is 10 nt. Another increase in affinity seen as the oligo(U) reached 24 nt suggests that a 24-nucleotide RNA can be occupied by two polymerase molecules. Direct binding of wild-type polymerase to oligo(U)12 and oligo(U)24 RNAs showed differences in affinity and cooperativity consistent with this model. The increase in binding affinity seen for oligo(U)10 suggests either that the RNA-binding determinants are widely spaced on the polymerase structure or that a substantial conformational change in the polymerase occurs upon the filling of its RNA-binding site.

Binding Sites↗
PubMed2003

Is the Trypanosoma brucei REL1 RNA ligase specific for U-deletion RNA editing, and is the REL2 RNA ligase specific for U-insertion editing?

It was shown previously that the REL1 mitochondrial RNA ligase in Trypanosoma brucei was a vital gene and disruption affected RNA editing in vivo, whereas the REL2 RNA ligase gene could be down-regulated with no effect on cell growth or on RNA editing. We performed down-regulation of REL1 in procyclic T. brucei (midgut insect forms) by RNA interference and found a 40-50% inhibition of Cyb editing, which has only U-insertions, as well as a similar inhibition of ND7 editing, which has both U-insertions and U-deletions. In addition, both U-insertion and U-deletion in vitro pre-cleaved editing were inhibited to similar extents. We also found little if any effect of REL1 down-regulation on the sedimentation coefficient or abundance of the RNA ligase-containing L-complex (Aphasizhev, R., Aphasizheva, I., Nelson, R. E., Gao, G., Simpson, A. M., Kang, X., Falick, A. M., Sbicego, S., and Simpson, L. (2003) EMBO J. 22, 913-924), suggesting that the inhibition of both insertion and deletion editing was not due to a disruption of the L-complex. Together with the evidence that down-regulation of REL2 has no effect on cell growth or on RNA editing in vivo or in vitro, these data suggest that the REL1 RNA ligase may be active in vivo in both U-insertion and U-deletion editing. The in vivo biological role of REL2 remains obscure.

Animals↗
PubMed2004

A relaxed discrimination of 2'-O-methyl-GTP relative to GTP between de novo and Elongative RNA synthesis by the hepatitis C RNA-dependent RNA polymerase NS5B.

Several nucleotide analogues have been described as inhibitors of NS5B, the essential viral RNA-dependent RNA polymerase of hepatitis C virus. However, their precise mode of action remains poorly defined at the molecular level, much like the different steps of de novo initiation of viral RNA synthesis. Here, we show that before elongation, de novo RNA synthesis is made of at least two distinct kinetic phases, the creation of the first phosphodiester bond being the most efficient nucleotide incorporation event. We have studied 2'-O-methyl-GTP as an inhibitor of NS5B-directed RNA synthesis. As a nucleotide competitor of GTP in RNA synthesis, 2'-O-methyl-GTP is able to act as a chain terminator and inhibit RNA synthesis. Relative to GTP, we find that this analogue is strongly discriminated against at the initiation step ( approximately 150-fold) compared with approximately 2-fold at the elongation step. Interestingly, discrimination of the 2'-O-methyl-GTP at initiation is suppressed in a variant NS5B deleted in a subdomain critical for initiation (the "flap," encompassing amino acids 443-454), but not in P495L NS5B, which shows a selective alteration of transition from initiation to elongation. Our results demonstrate that the conformational change occurring between initiation and elongation is dependent on the allosteric GTP-binding site and relaxes nucleotide selectivity. RNA elongation may represent the most probable target of 2'-modified nucleotide analogues, because it is more permissive to inhibition than initiation.

Allosteric Site↗
PubMed2001

RNA-dependent RNA polymerase activity associated with endogenous double-stranded RNA in rice.

RNA-dependent RNA polymerase (RdRp) activity was detected in the crude microsomal fraction of rice cultured cells that contain a 14 kbp double-stranded RNA (dsRNA). RdRp activity is maximal in the presence of all four nucleotide triphosphates and Mg2+ ion and is resistant to inhibitors of DNA-dependent RNA polymerases (actinomycin D and alpha-amanitin). RdRp activity increases approximately 2.5-fold in the presence of 0.5% deoxycholate. Treatment of purified microsomal fraction with proteinase K plus deoxycholate suggests that the RdRp enzyme complex with its own 14 kb RNA template is located in vesicles. The RdRp enzyme complex was solubilized with Nonidet P-40 and purified by glycerol gradient centrifugation, then exogenous RNA templates were added. Results indicate that exogenous dsRNA reduces RNA synthesis from the endogenous 14 kb RNA template.

Cells, Cultured↗
PubMed1996

Influenza A virus RNA-dependent RNA polymerase: analysis of RNA synthesis in vitro.

Influenza A virus RNA-dependent RNA polymerase, purified from virion ribonucleoprotein particles and from which endogenous genomic RNA (vRNA) has been depleted by treatment with micrococcal nuclease, was used to study transcription initiation, elongation, and termination in vitro. Templates that contained either minus- or plus-sense influenza virus nucleoprotein minigenes with conserved 5' and 3' termini and the uridylate tract were constructed. The dinucleotide ApG and alfalfa mosaic virus RNA4 (AlMV4) were used as primers. ApG primed the synthesis of full-length positive-strand or cRNA products and shorter transcripts, depending upon the molar ratio between the nucleoprotein and the vRNA template. Sequence analysis of the ends of these transcripts demonstrated that the 5' termini of both transcripts and the 3' terminus of the full-length product were complementary to the 3' and 5' termini of the vRNA template, respectively, whereas the 3' terminus of the incomplete product corresponded to a sequence located 40 bases downstream from the 5' terminus of the template and was about 20 nucleotides downstream from the uridylate tract, which is the putative signal for polyadenylation. Binding of the cap structure of AlMV4 by the polymerase activated RNA synthesis by ligation-elongation of small genomic RNA fragments which were likely derived from a genome segment protected by the polymerase from micrococcal nuclease digestion. The sequence of these fragments mapped to a region 14 to 28 nucleotides upstream of the 3' terminus of the viral genome. Polymerase subunit involvement in transcription initiation with ApG or AlMV4 was characterized by studying the effect of purified polyclonal antisubunit immunoglobulins of the G class (IgGs) in transcription assays. These results showed that anti-PB2 IgG inhibited transcription initiation in both ApG- and AlMV4-primed reactions, whereas anti-PB1 antibodies also blocked transcription initiated with AlMV4. The differences observed in product size, product sequence, and differential inhibition by antisubunit IgGs are discussed. These observations would support the notion that the influenza virus RNA-dependent RNA polymerase undergoes a conformational change after the binding of the cap structure of host cell heterogeneous nuclear RNA by PB2, which then usually leads to endonucleolytic cleavage of the capped primer 13 nucleotides downstream from the cap.

Animals↗