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1.
The RNA products synthesized by the virion-associated RNA polymerase of purified vesicular stomatitis virus have previously been shown to contain two distinct 5′-terminal sequences. The mRNA species contain the blocked 5′-terminal G(5′)ppp(5′)A-A-C-A-G sequence and the initiated lead-in RNA segment (approximately 50 bases) contains the unblocked 5′ ppA-C-G sequence. In the present studies, using inosine 5′-triphosphate in place of GTP it is shown that RNA species as large as 14.5S contain an unblocked 5′-ppA-C-(I) sequence indicating that the GTP analogue permits synthesis of a possible precursor of viral mRNA . 相似文献
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Several plant and animal viral RNAs contain a tRNA like structure at their 3′ ends. In this communication we show that tobacco mosaic virus (TMV) RNA is an acceptable substrate for a specific tRNA methyltransferase. Using a crude preparation of ribothymidine (rT) forming uracil methylase and (methyl 3H) S-adenosyl-L-methionine (SAM) as a methyl donor, 0.7 moles of methyl group is incorporated per mole of TMV RNA in 10 hours at 30°C. Upon T2 RNAse digestion of the labeled RNA, all of the radioactivity was found to be in TMP. T1 RNAse digestion of 3H methylated TMV RNA showed that all of the label was located in a tetranucleotide which co-migrated with authentic TpψpCpGp, an oligonucleotide characteristically found in normal cellular tRNA.The use of this specific methyl transferase reaction may provide a simple assay for the detection of tRNA like structures in large RNAs. 相似文献
5.
The broad spectrum antiviral agent ribavirin inhibits capping of mRNA. 总被引:14,自引:0,他引:14
B B Goswami E Borek O K Sharma J Fujitaki R A Smith 《Biochemical and biophysical research communications》1979,89(3):830-836
Ribavirin (1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a broad spectrum antiviral substance active against a wide range of both DNA and RNA viruses. It is, however, virtually inactive against polio virus. Its pharmacological mechanism of action was obscure. A possible common target for a chemotherapeutic agent in both DNA and RNA viruses is the “capping” reaction of mRNAs which involves the formation of a guanine pyrophosphate structure at the 5′ terminus by mRNA guanylyl transferase. We have observed that Ribavirin triphosphate is a potent competitive inhibitor of the capping guanylation of viral mRNA. This finding could account for the antiviral potency of the drug against both DNA and RNA viruses and its ineffectiveness against a virus in which the mRNAs derived from them are not capped. 相似文献
6.
The distribution of binding sites for R-MuLV p12 phosphoprotein on the viral genome has been examined. Ribonucleoprotein complexes formed using 3′-poly A-containing viral RNA fragments of varying lengths and radioiodinated p12 protein have been analyzed by sedimentation velocity and buoyant density gradients. Binding sites for 2–3 molecules of p12 protein can be detected within the first 400 nucleotides from the 3′-poly A segment. The possible presence of binding sites near the middle of the genome (~2500 nucleotides from the 3′-end) and very close to the 5′-terminus (within the terminal 100–200 nucleotides) is also indicated. 相似文献
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J Germershausen D Goodman E W Somberg 《Biochemical and biophysical research communications》1978,82(3):871-878
RNA (guanine-7) methyltransferase, partially purified from mycelia, catalyzed the transfer of the methyl group from S-adenosylmethionine to the 5′ terminus of both poly A(+) RNA and reovirus unmethylated mRNA. RNase T2 digestion of the methylated poly A(+) RNA from yielded the “cap” structures m 7G(5′)pppAp and m 7G(5′)pppGp in a ratio of 2:1 respectively. RNase T2 digestion of the methylated reovirus mRNA yielded m 7G(5′)pppGp exclusively. The absence of mRNA 2′-0-methyltransferase activity in the enzyme preparation is consistent with the absence of 2′-0-methylation in mRNA [Seidel, B. L. and Somberg, E. W. (1978) Arch. Biochem. Biophys. , 108–112]. This is the first isolation of an eucaryotic, cellular RNA (guanine-7) methyltransferase that has been shown to methylate homologous substrate. 相似文献
8.
A Stevens 《Biochemical and biophysical research communications》1978,81(2):656-661
Using poly(A) as a substrate, an exoribonuclease has been purified from the high-salt wash of ribosomes of . The product of the reaction of the exoribonuclease is 5′ AMP. Hydrolysis of [3H](pA)3[14C](pA)n shows that both labels are released at the same rate, suggesting that the enzyme acts in a processive manner. Removal of the terminal phosphate of poly(A) with alkaline phosphatase reduces the rate of hydrolysis by 80%. Treatment of the terminally dephosphorylated poly(A) with polynucleotide kinase restores the activity. Two 5′ capped mRNA's have been tested and they are hydrolyzed slowly, if at all, by the enzyme. In contrast, phage T4 mRNA, ribosomal RNA, and encephalomyocarditis viral RNA are hydrolyzed at greater than 50% of the rate of poly(A). 相似文献
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In vitro association of unique species of cellular 4S RNA with 35S RNA of RNA tumor viruses 总被引:3,自引:0,他引:3
L C Waters B C Mullin T Ho W K Yang 《Biochemical and biophysical research communications》1974,60(2):489-497
Unique 4S RNA species from AKR mouse embryo cells hybridize with AKR murine leukemia virus and avian myeloblastosis virus 35S RNAs . Analyses by reversed-phase column chromatography indicate that the major 4S species that hybridize with the two viral RNAs are probably the same. A 4S RNA species with similar chromatographic properties is a major component of the AKR viral 4S RNA which associates with the viral 70S RNA . 相似文献
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Methylation of adenovirus 2 (Ad 2) late RNA was studied. RNA was double-labeled with [3H-methyl]-methionine and [14C]-uridine 15–20 h postinfection. Nuclear RNA (rRNA) and cytoplasmic RNA (mRNA) was extracted, and fractionated into polyA(+) and (?) molecules using poly(U)-Sepharose. Ad 2 specific RNA was purified by 2 cycles of hybridization to and elution from Ad 2 DNA immobilized on filters. The Ad 2 polyA(+) and (?) nRNA and mRNA fractions had the same ratios, and were estimated to contain a minimum of 1.4 methylated nucleotides per 1000 bases. Viral RNA was digested with RNase T2 and chromatographed on DEAE-Sephadex in 7 M urea at pH 7.6. All four Ad RNA fractions contained methylated constituents consistent with: (1) two classes of methylated “capped” 5′-termini with general structures m7 GpppNmpNp and m7 GpppNmpNmpNp; (2) internal base methylations; (3) minor amounts of internal ribose 2′-0-methylations. Two classes of 5′-termini have previously been reported for animal cell mRNA, but not for mRNA from a variety of viruses. Internal methylations may be unique to RNA molecules transcribed in the nucleus, since they have not been found in RNA from cytoplasmic viruses. No gross differences were observed in the DEAE-Sephadex elution profiles of the methylated constituents of the four types of Ad 2 RNA. These results suggest that the majority of methylation events occur in the nucleus, and raise the possibility that Ad 2 methylated late nRNA may differ significantly from SV40 late nRNA (Lavi, S., and Shatkin, A.J. (1975) Proc. Natl. Acad. Sci. USA , 2012–2016). 相似文献
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S N Seal A Schmidt M Tomaszewski A Marcus 《Biochemical and biophysical research communications》1978,82(2):553-559
The cap analogue, 7-methylguanosine-5′-phosphate (pm7G), inhibits the translation of the noncapped STNV (satellite tobacco necrosis virus) RNA and CPMV (cowpea mosaic virus) RNA in the wheat germ protein synthesizing system. While the translation of some capped mRNAs is inhibited more strongly by the analogue, other capped mRNAs have a level of sensitivity similar to that of the noncapped RNAs. Evidence is presented demonstrating that the effect of the analogue is exerted at a cap binding site even when it is inhibiting noncapped mRNAs. These results therefore indicate that the cap binding site of the translational system is either part of or is closely linked to another mRNA binding component, this component being specific for a site on the mRNA other than the 5′ cap. The observations also suggest caution in the use of pm7G inhibition to indicate the presence of a 5′ cap on a particular mRNA. 相似文献
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Thermal denaturation of nucleocapsids of wild type (WT) vesicular stomatitis virus (VSV), containing only the nucleocapsid protein (N) and viral RNA, caused a “melting” that resulted in an A260nm absorbance increase of 140%. The nucleocapsids of two temperature-sensitive () VSV mutants, G31BP and G22, both underwent larger absorbance increases of 251% and 177% respectively, suggesting these nucleocapsids are complexed by weaker N protein: RNA interactions than the WT-VSV. Two other mutants, G31 and G41 underwent A260nm increases either similar to, or smaller than, that measured with WT-VSV nucleocapsids. RNA synthesis by G31BP in infected cells was also found to be decreased at elevated temperatures. This temperature sensitive defect in viral RNA metabolism in G31BP may be the result of weaker protein:RNA interactions associated with the nucleocapsid. 相似文献
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Cell-free extracts of seedlings exhibited enzyme activities for the following reactions: S-adenosyl-L-methionine (SAM) decarboxylation, spermidine synthesis from decarboxylated SAM and putrescine, and 5′-methylthioadenosine hydrolysis to 5-S-methyl-5-thio-D-ribose and adenine. SAM decarboxylation was stimulated by putrescine and inhibited by semicarbazide. The 15-fold purified ribohydrolase possessed a Km of 1. 03 × 10?5 M and a high specificity for 5′-methylthioadenosine. 相似文献
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Transcription of chromatin by human RNA polymerase 总被引:3,自引:0,他引:3
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Comparison of methylated sequences in messenger RNA and heterogeneous nuclear RNA from mouse L cells 总被引:9,自引:0,他引:9
A variety of methylated oligonucleotides were derived from mouse L cell messenger RNA and heterogeneous nuclear RNA by digestion with specific ribonucleases, and the cap-containing oligonucleotides separated from those containing internal m6A by chromatography on diborylaminoethyl-cellulose. Cap-containing sequences of the type m7GpppXmpG, m7GpppXmpY(m)pG, m7GpppXmpY(m) pNpG and m7GpppXmpY(m)p(Np)> 1G have distinctive non-random compositions of the 2′-O-methylated constituent Xm; yet sequences of a particular type and composition occur with a remarkably similar frequency in mRNA and hnRNA2. For example, approximately 20% of the cap sequences in both hnRNA and mRNA are m7Gppp(m6)AmG, whereas less than 1% are m7GpppUmpG. The high degree of similarity in cap sequences is consistent with the previously postulated precursor-product relationship between hnRNA caps and mRNA caps.The composition of the Y position in capped hnRNA molecules was determined to be (29% G, 20% A, 51% Py), which differs considerably from the composition of Ym in the cap II forms of mRNA (8% Gm, 11% Am, 81% Py). Given the precursor-product relationship between hnRNA caps and mRNA caps, this result provides strong evidence that only a restricted subclass of mRNA molecules receive the secondary methylation at position Y.In both hnRNA and mRNA the internal m6A occurs in well-defined sequences of the type: , the 5′ nearest-neighbor of m6A being G in about three-quarters of the molecules and A in about one-quarter of the molecules. The nucleotide N1 is a purine about 90% of the time and the nucleotide N2 is rarely a G. These same sequences are present in large (> 50 S), as well as small (14 S to 50 S) hnRNA. These results raise the possibility that the internal m6A, like caps, may be conserved during the processing of large hnRNA into mRNA. Two models based on this idea are discussed. 相似文献
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N-6-methyl-adenosine in adenovirus type 2 nuclear RNA is conserved in the formation of messenger RNA 总被引:18,自引:0,他引:18
In the biogenesis of adenovirus type 2 messenger RNAs, methylation occurs at the 5′ end (cap) and to internal adenosine residues to yield N6-methyl-adenosine (m6A) (Sommer et al., 1976; Moss &; Koczot, 1976; Wold et al., 1976). The kinetics of accumulation of 3H from methyl-labeled methionine and 14C from uridine into Ad-22-specific RNA was measured late in Ad-2 infection. As reported previously (Nevins &; Darnell, 1978a), the rate of accumulation of [14C]uridine label in nuclear RNA was approximately four- to fivefold faster than in the cytoplasmic RNA, indicating a conservation of about 20% for the total RNA. The initial rates of [3H]methyl label in m6A in nuclear RNA and in the cytoplasmic RNA were approximately equal, suggesting a complete (or nearly complete) conservation of m6A.In accord with the accumulation kinetics, the ratio of 3H to 14C was higher in cytoplasmic RNA than in nuclear RNA that hybridized to equivalent regions of the Ad-2 DNA.A mathematical model was designed to evaluate the accumulation of methyl label in m6A, taking into consideration the three major parameters that affect the accumulation curves: equilibration of the S-adenosyl-methionine pool, the nuclear dwell time of sequences destined to be mRNA, and the cytoplasmic stability of mRNA. The half-time () for pool equilibration was determined experimentally to be 22 minutes and the nuclear dwell time and the mean life-time of cytoplasmic mRNA were estimated from 14C label to be about 30 and 70 minutes, respectively.The model gave an excellent fit to the data when the for pool equilibration time of 24 ± 2 minutes, a nuclear dwell time of 25 ± 10 minutes, and a mean cytoplasmic mRNA life-time of 75 ± 30 minutes were used to evaluate accumulation curves. Even when data from a restricted region of the genome, , which encodes the main portion of at least five 3′ co-terminal mRNAs whose spliced junction with the tripartite leader sequence varies from , and was analyzed, it appeared that m6A was conserved.Finally, m6A was found to be added in a brief label (3.5 min) mainly to nuclear molecules that were longer than any cytoplasmic RNA. The conservation of m6A and its addition prior to splicing raise the possibility that internal methylations are involved, in the formation of mRNA. 相似文献
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Initiator RNA of nascent DNA from animal cells. 总被引:3,自引:0,他引:3
Nascent DNA synthesized by intact cells has been examined for the presence of RNA that may function as a primer in the discontinuous synthesis of DNA. A low molecular weight fraction that contains nascent DNA was isolated from a human lymphoblastoid cell line in logarithmic growth. After labeling the 5′ ends with bacteriophage T4 polynucleotide kinase and [γ-32P]ATP, and digestion of the DNA with DNAase, a DNAase-resistant oligonucleotide was isolated. This fragment consisted of approximately 9 ribonucleotide residues, with 5′ terminal purines (), plus one to three 3′ terminal deoxynucleotides resulting from incomplete removal by DNAase. Approximately 10% of short nascent DNA chains contained the nonanucleotide molecule. An additional 20% of the nascent DNA contained ribooligomers shorter than 9 residues, with 5′ termini substantially increased in pyrimidines, which may result from degradation of the nonanucleotide. These results extend previous studies that demonstrated a similar ribooligonucleotide present at the 5′ end of most or all short nascent DNA chains synthesized in broken cell systems. Together with the results obtained by Reichard and co-workers (Reichard et al., 1974) with polyoma virus, the data support a mechanism by which a short initiator RNA serves as primer for discontinuously synthesized DNA in animal cells. 相似文献
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Maria Szekely Therese Loviny Richard C. Foreman 《Biochemical and biophysical research communications》1976,70(1):110-116
Binding of formylmethionyl tRNA and ribosomes to double stranded RNA has been obtained under conditions identical to those required for initiation complex formation with single stranded RNA. While natural double stranded RNAs from Penicillium virus and Penicillium virus were efficient in forming initiation complexes, the synthetic polynucleotide poly(I).poly (C) was inactive. This suggests that ribosomes can recognize initiation sequences even if these are present in base-paired form. 相似文献
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Structures at the 5′ terminus of poly (A)-containing cytoplasmic RNA and heterogeneous nuclear RNA containing and lacking poly(A) have been examined in RNA extracted from both normal and heat-shocked Drosophila cells. 32P-labeled RNA was digested with ribonucleases T2, T1 and A and the products fractionated by a fingerprinting procedure which separates both unblocked 5′ phosphorylated termini and the blocked, methylated, “capped” termini, known to be present in the messenger RNA of most eukaryotes.Approximately 80% of the 5′-terminal structures recovered from digests of poly(A)-containing Drosophila mRNA are cap structures of the general form m7G5′ppp5′X(m)pY(m)pZp. With respect to the extent of ribose methylation and the base distribution, the 5′-terminal sequences of Drosophila capped mRNA appear to be intermediate between those of unicellular eukaryotes and those of mammals. Drosophila is the first organism known in which type 0 (no ribose methylations), type 1 (one ribose methylation), and type 2 (two ribose methylations) caps are all present. In contrast to mammalian cells, the caps of Drosophila never contain the doubly methylated nucleoside N6,2′-O-dimethyladenosine. Both purines and pyrimidines can be found as the penultimate nucleoside of Drosophila caps and there is a wide variety of X-Y base combinations. The relative frequencies of these different base combinations, and the extent of ribose methylation, vary with the duration of labeling. The large majority of poly(A)-containing cytoplasmic RNA molecules from heat-shocked Drosophila cells are also capped, but these caps are unusual in having almost exclusively purines as the penultimate X base.Greater than 75% of the 5′ termini of heterogeneous nuclear RNA (hnRNA) containing poly(A) and greater than 50% of the termini of hnRNA lacking poly (A) are also capped. Triphosphorylated nucleotides, common as the 5′ nucleotides of mammalian hnRNA, are rare in the poly(A)-containing hnRNA of Drosophila. The frequency of the various type 0 and type 1 cap sequences of cytoplasmic and nuclear poly (A)-containing RNA are almost identical. The caps of hnRNA lacking poly(A) are also quite similar to those of poly-adenylated hnRNA, but are somewhat lower in their content of penultimate pyrimidine nucleosides, suggesting that these two populations of molecules are not identical. 相似文献