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PubMed1977

Characterization and in vitro translation of polyadenylated messenger ribonucleic acid from Neurospora crassa.

Ribonucleic acid (RNA) extracted from Neurospora crassa has been fractionated by oligodeoxythymidylic acid [oligo(dT)]-cellulose chromatography into polyadenylated messenger RNA [poly(A) mRNA] and unbound RNA. The poly(A) mRNA, which comprises approximately 1.7% of the total cellular RNA, was further characterized by Sepharose 4B chromatography and polyacrylamide gel electrophoresis. Both techniques showed that the poly(A) mRNA was heterodisperse in size, with an average molecular weight similar to that of 17S ribosomal RNA (rRNA). The poly(A) segments isolated from the poly(A) mRNA were relatively short, with three major size classes of 30, 55, and 70 nucleotides. Gel electrophoresis of the non-poly(A) RNA indicated that it contained primarily rRNA and 4S RNA. The optimal conditions were determined for the translation of Neurospora mRNA in a cell-free wheat germ protein-synthesizing system. Poly(A) mRNA stimulated the incorporation of [14C]leucine into polypeptides ranging in size from 10,000 to 100,000 daltons. The RNA that did not bind to oligo(dT)-cellulose also stimulated the incorporation of [14C]leucine, indicating that this fraction contains a significant concentration of mRNA which has either no poly(A) or very short poly(A) segments. In addition, the translation of both poly(A) mRNA and unbound mRNA was inhibited by 7-methylguanosine-5'-monophosphate (m7G5'p). This is preliminary evidence for the existence of a 5'-RNA "cap" on Neurospora mRNA.

Chromatography, Agarose↗

PubMed1978

mRNA(nucleoside-2'-)-methyltransferase from vaccinia virus. Characteristics and substrate specificity.

An mRNA(nucleoside-2'-)-methyltransferase purified from vaccinia virus was shown to methylate the penultimate nucleoside of RNA ending in m7G(5')pppN-. By contrast, RNAs ending in pN-, ppN-, or even G(5')pppN- are not methyl acceptors. This specificity indicates that 2'-O-methylation is the final step in the formation of the m7G(5')pppNm- cap structure. Both adenosine and guanosine are methylated, in accordance with the presence of these nucleosides in the penultimate position of vaccinia virus mRNAs. Studies with homopolyribonucleotides containing m7G(5')pppN ends indicated that poly(A) and poly(I) were the best methyl acceptors while significant but much less activity was obtained with poly(G), poly(U), and poly(C). Simple dinucleotides of the type m7G(5')pppN, however, are poor substrates and do not compete with capped RNA. Additional studies indicate that the methyltransferase has a pH optimum of 7.5, does not require divalent cations, is inhibited by S-adenosylhomocysteine, has a Km of 2.0 micrometer for S-adenosylmethionine, a Km of approximately 5 nM for brome mosaic virus RNA, and kinetics consistent with a random bireactant mechanism.

Kinetics↗

PubMed2005

Structure of the Flavivirus helicase: implications for catalytic activity, protein interactions, and proteolytic processing.

Yellow fever virus (YFV), a member of the Flavivirus genus, has a plus-sense RNA genome encoding a single polyprotein. Viral protein NS3 includes a protease and a helicase that are essential to virus replication and to RNA capping. The 1.8-A crystal structure of the helicase region of the YFV NS3 protein includes residues 187 to 623. Two familiar helicase domains bind nucleotide in a triphosphate pocket without base recognition, providing a site for nonspecific hydrolysis of nucleoside triphosphates and RNA triphosphate. The third, C-terminal domain has a unique structure and is proposed to function in RNA and protein recognition. The organization of the three domains indicates that cleavage of the viral polyprotein NS3-NS4A junction occurs in trans.

Adenosine Triphosphatases↗

PubMed1998

Brome mosaic virus RNA replication protein 1a dramatically increases in vivo stability but not translation of viral genomic RNA3.

Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two RNA replication proteins: 1a, which contains a helicase-like domain and a domain implicated in RNA capping, and 2a, which contains a polymerase-like domain. To further explore their functions, we expressed 1a and 2a individually and together in yeast also expressing replicatable transcripts of BMV genomic RNA3. Complementing prior results that 1a and 2a are required jointly for positive-strand RNA synthesis, both also were required for negative-strand RNA synthesis. Nevertheless, in the absence of 2a, 1a expression increased the accumulation of DNA-derived RNA3 transcripts 8-fold. Increased accumulation was specific for RNA3: none of a diverse set of yeast mRNAs tested showed increased accumulation in the presence of 1a. Increased RNA3 accumulation was not due to increased DNA transcription, but to a 20- to 40-fold increase in the in vivo half-life of RNA3 from 5-10 min in the absence of 1a to more than 3 hr in the presence of 1a. Although (1a+2a)-dependent RNA replication selectively amplified the natural viral 5' end from among multiple transcription starts of DNA-derived RNA3 transcripts, 1a-induced stabilization affected all RNA3 transcripts, without specificity for the precise viral 5' end. Increased RNA3 accumulation did not increase expression of a directly translatable, 5'-proximal gene in RNA3, implying that 1a-induced stabilization blocked rather than facilitated RNA3 translation. These and other results suggest that the striking, 1a-induced stabilization of RNA3 may reflect an interaction involved in recruiting viral RNA templates into RNA replication while diverting them from the competing processes of translation and degradation.

Bromovirus↗

PubMed1986

Activation of the AIDS retrovirus promoter by the cellular transcription factor, Sp1.

The nature and position of transcriptional control elements responsible for the expression of genes encoded by the retrovirus associated with acquired immune deficiency syndrome (AIDS) have not been precisely defined. In this study it is shown that the mammalian Sp1 transcription factor binds to promoter sequences within the AIDS retrovirus long terminal repeat (LTR) and activates RNA synthesis five- to eightfold in reconstituted reactions in vitro. Experiments in which regions of DNA were protected from added reagents by specifically bound proteins (footprinting) indicated that the upstream promoter region of the AIDS virus LTR lies between -45 and -77 (relative to the RNA start site, +1) and contains three tandem, closely spaced SP1 binding sites of variable affinity. Base-substitution mutations targeted to one or all three Sp1 binding sites were found both to eliminate the binding of Sp1 and to cause up to a tenfold reduction in transcriptional efficiency in vitro. These findings suggest that one important component of the AIDS virus transcriptional control region interacts with a cellular transcription factor, Sp1, and that this factor must function in conjunction with transcriptional elements located downstream of the RNA cap site to mediate the response of the LTR to viral trans-activation.

Acquired Immunodeficiency Syndrome↗

PubMed1991

Sequence-independent RNA cleavages generate the primers for plus strand DNA synthesis in hepatitis B viruses: implications for other reverse transcribing elements.

Reverse transcription of RNA into duplex DNA requires accurate initiation of both minus and plus strand DNA synthesis; this in turn requires the generation of specific primer molecules. We have examined plus strand primer generation in the hepatitis B viruses, small DNA viruses that replicate via reverse transcription. The plus strand primer in these viruses is a short capped RNA derived from the 5' end of the RNA template by cleavage at a specific set of sites. To elucidate the cleavage mechanism we constructed a series of viral mutants bearing alterations in and around the cleavage sites. Our results reveal that the cleavage reaction is sequence-independent and indicate that the cleavage sites are positioned by measurement of the distance from the 5' end of the RNA. Comparison of these findings with what is known about RNase H-mediated primer generation in retroviruses and other retroid elements suggests that, despite many divergent features, some common molecular features are preserved.

Base Sequence↗

PubMed1997

Versatile 5' phosphoryl coupling of small and large molecules to an RNA.

A Ca2+-requiring catalytic RNA is shown to create 5' phosphate-phosphate linkages with all nucleotides and coenzymes including CoA, nicotinamide adenine dinucleotide phosphate, thiamine phosphate, thiamine pyrophosphate, and flavin mononucleotide. In addition to these small molecules, macromolecules such as RNAs with 5'-diphosphates, and nonnucleotide molecules like Nepsilon-phosphate arginine and 6-phosphate gluconic acid also react. That is, the self-capping RNA isolate 6 is an apparently universal 5' phosphate-linker, reacting with any nucleophile containing an unblocked phosphate. These RNA reactions demonstrate a unique RNA catalytic capability and imply versatile and specific posttranscriptional RNA modification by RNA catalysis.

Calcium↗

PubMed1982

Sequence analysis of the polymerase 1 gene and the secondary structure prediction of polymerase 1 protein of human influenza virus A/WSN/33.

The nucleotide sequence of polymerase 1 (P1) gene of a human influenza virus (A/WSN/33) has been determined by using cDNA clones, except for the last 83 nucleotides, which were obtained by primer extension. The WSN P1 gene contains 2,341 nucleotides and codes for a protein of 757 amino acids (Mr = 86,500). P1 gene possesses a striking tandem repeat of 12 nucleotides (nucleotide position 2,188 to 2,199, 2,200 to 2,211) and a corresponding tandem repeat of tetrapeptide in the P1 protein. The deduced sequence of P1 protein is enriched in basic amino acids, particularly arginine. In addition, it also contains clusters of basic amino acids which may provide sites for the interaction with the template virion RNA capped primer as well as with other proteins involved in viral replication and transcription. A secondary structure prediction, using Chou and Fasman analyses (Annu. Rev. Biochem. 47:251-276, 1978), shows that the P1 protein possesses some unique features, viz., one "four-helical supersecondary structure" and four "polypeptide double helices" (antiparallel beta-pleated sheets) which are considered important in RNA binding.

Amino Acid Sequence↗

PubMed1982

A nonanucleotide sequence involved in promotion of ribosomal RNA synthesis and RNA priming of DNA replication in yeast mitochondria.

We have examined the initiation of transcription of the mitochondrial genes for ribosomal RNA (rRNA) in the yeast Kluyveromyces lactis and show that these are transcribed independently from individual promoters. The mature large rRNA contains a 5' di- or triphosphate end which can be labelled in vitro with [alpha-32P]GTP using guanylyltransferase and this enabled us to determine the nucleotide sequence of its 5' terminus. For the small rRNA, a minor in vitro capped RNA species hybridizes in the region where--as judged from S1 nuclease protection experiments--the precursor of this RNA starts. We have determined the DNA sequence around the beginning of both rRNA genes and this reveals the existence of an identical nonanucleotide sequence (5' -ATATAAGTA- 3') just preceding the positions where the rRNAs start. This sequence is identical to the one preceding the rRNA genes in the mtDNA of the distantly related yeast Saccharomyces cerevisiae (Osinga, K.A. and Tabak, H.F. (1982) Nucl.Acids Res. 10, 3617-3626) and supports our proposal that this sequence motif is part of a yeast mitochondrial promoter. We have noticed that the same sequence is located in the putative origin of replication present in hypersuppressive petite mutants of S. cerevisiae and consider the possibility that this sequence is involved in RNA priming of DNA replication.

Ascomycota↗

PubMed1983

Identification of transcriptional elements within the long terminal repeat of Rous sarcoma virus.

Transcriptional regulatory elements within the Rous sarcoma virus long terminal repeat were examined by the construction of a series of deletions and small insertions within the U3 region of the long terminal repeat. The analysis of these mutations in chicken embryo cells and COS cells permitted the identification of important transcriptional regulatory elements. Sequences within the region 31 to 18 base pairs upstream of the RNA cap site (-31 to -18), encompassing a TATA box-like sequence, function in the selection of the correct site of transcription initiation and, in addition, augment the efficiency of transcription. These sequences are essential for virus replication. Sequences within the region -79 to -59, overlapping a CAAT box-like sequence, are not required for virus replication and have no obvious effect on viral RNA transcription in the presence of an intact TATA box. However, in mutants lacking a functional TATA sequence, mutations in this region serve to decrease the efficiency of correct transcriptional initiation events.

Animals↗

PubMed1995

Identification and analysis of the start site of ribosomal RNA transcription of Entamoeba histolytica.

In this article we report the identification of the start site of ribosomal RNA transcription unit of the enteric parasite E. histolytica. We cloned the upstream region of the ribosomal RNA and we defined the 5' boundary of the transcription unit with nuclear run-on assays. We report that ribosomal transcription starts 2447 bp upstream the SSU ribosomal gene, at an adenosine residue. This data was supported both by S1 mapping and by primer extension analysis; that the mapped site was indeed the transcription start point was demonstrated by RNAse protection of the in vitro capped RNA. Our sequence data around the transcription start point shows two different tandem repeat clusters immediately downstream from the transcription start point.

Animals↗

PubMed1989

SVLM21, a Sindbis virus mutant resistant to methionine deprivation, encodes an altered methyltransferase.

The replication of Sindbis virus (SVSTD) in cultured Aedes albopictus mosquito cells is sensitive to methionine deprivation. We have suggested from earlier work that this sensitivity is primarily because of a decreased pool of S-adenosyl methionine (ado met) and the resultant failure to methylate the 5' cap of the viral mRNAs. SVLM21, a strain of Sindbis virus derived in our laboratory from SVSTD by serial passage on mosquito cells maintained after infection in low concentrations of methionine, is resistant to methionine starvation. It was proposed that this adaptation to low methionine, and to the resultant low intracellular levels of ado met, reflected the accumulation of mutations which led to the generation of a viral RNA cap methyltransferase with an increased affinity for ado met. We report here kinetic data which distinguished the enzymes coded for by SVSTD and SVLM21. Using guanylylimidodiphosphate (GIDP) as the methyl acceptor, radioactively labeled ado met as the methyl donor, and lysates from infected BHK cells as the enzyme source, we calculated from our results that SVLM21 generated a methyltransferase with a Km for ado met 10-fold lower than that generated by either SVSTD or the related alphavirus, Semliki Forest virus. In addition, we found that BHK cells infected with SVLM21 generated higher levels of methyltransferase activity than did cells infected with SVSTD and that the SVSTD and SVLM21 enzymes differed with respect to their relative activities at elevated temperatures. We conclude from these results that the SVLM21 phenotype is associated with an altered methyltransferase and suggest that this is the basis of the resistance of SVLM21 to methionine deprivation.

Animals↗

PubMed1982

Reaction mechanism of mRNA guanylyltransferase from rat liver: isolation and characterization of a guanylyl-enzyme intermediate.

Rat liver RNA guanylyltransferase catalyzes a GTP-PPi exchange reaction in the absence of acceptor RNA [Mizumoto, K. & Lipmann, F. (1979) Proc. Natl. Acad. Sci. USA 76, 4961-4965] suggesting that the reaction proceeds through the formation of a covalent guanylylated intermediate. We now present more direct evidence for the existence of the enzyme-GMP intermediate: (i) the enzyme-[32P]GMP intermediate was formed on incubation of rat liver guanylyltransferase with [alpha-32P]GTP and migrated as a single radioactive band with Mr 69,000 on NaDodSO4/polyacrylamide gel electrophoresis, and (ii) the intermediate isolated on gel filtration can transfer its GMP moiety to ppGpCpC-poly(A2,U2,G) to form the capped RNA molecule or it can react with PPi to regenerate GTP. The formation of the intermediate was dependent on Mg2+ and was strongly inhibited by PPi. The addition of pyrophosphatase markedly increased the amount of the intermediate complex. On blue dextran-Sepharose affinity column chromatography, the activity of guanylyltransferase to form an enzyme-[32P]GMP intermediate comigrated with activities of cap formation and GTP-PPi exchange. A phosphoamide type linkage between GMP and enzyme is suggested by its acidlabile and alkali-stable nature and also by the susceptibility to acidic hydroxylamine. These results indicate that the reaction catalyzed by rat liver guanylyltransferase occurs through the following two partial steps: (i) E + GTP in equilibrium E-pG + PPi; and (ii) E-pG + ppN .....leads to GpppN .....+ E.

Animals↗

PubMed1999

A brome mosaic virus intergenic RNA3 replication signal functions with viral replication protein 1a to dramatically stabilize RNA in vivo.

Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two RNA replication proteins. The 1a protein has putative helicase and RNA-capping domains, whereas 2a contains a polymerase-like domain. Saccharomyces cerevisiae expressing 1a and 2a is capable of replicating a BMV RNA3 template produced by in vivo transcription of a DNA copy of RNA3. Although insufficient for RNA3 replication, the expression of 1a protein alone results in a dramatic and specific stabilization of the RNA3 template in yeast. As one step toward understanding 1a-induced stabilization of RNA3, the interactions involved, and its possible relation to RNA replication, we have identified the cis-acting sequences required for this effect. We find that 1a-induced stabilization is mediated by a 150- to 190-base segment of the RNA3 intergenic region corresponding to a previously identified enhancer of RNA3 replication. Moreover, this segment is sufficient to confer 1a-induced stability on a heterologous beta-globin RNA. Within this intergenic segment, partial deletions that inhibited 1a-induced stabilization in yeast expressing 1a alone resulted in parallel decreases in the levels of negative- and positive-strand RNA3 replication products in yeast expressing 1a and 2a. In particular, a small deletion encompassing a motif corresponding to the box B element of RNA polymerase III promoters dramatically reduced the ability of RNAs to respond to 1a or 1a and 2a. These and other findings suggest that 1a-induced stabilization likely reflects an early template selection step in BMV RNA replication.

Bromovirus↗

PubMed2001

Construction of infectious dengue 2 virus cDNA clones using high copy number plasmid.

Procedures for cloning entire dengue serotype 2 virus genome in the multiple cloning site of a commercially available high copy number plasmid are described. The 10.7 kb viral RNA genome was reverse transcribed, amplified as three overlapping DNA fragments and successively ligated into pBluescript II KS, which contains the colE1 origin of replication. When propagated at room temperature (20-25 degrees C) under low level of antibiotic selection, the full-length recombinant plasmid was stable upon serial passages in two common Escherichia coli strains employed. Under the same culture conditions the whole dengue cDNA sequence was transferred successfully to another high copy number plasmid, pGem 3Z. Following in vitro transcription and lipofectin-mediated transfection, capped RNA transcripts derived from the plasmid initiated virus replication in C6/36 mosquito cells and BHK-21 cells within 3-4 days of transfection. Upon subsequent expansion in C6/36 cells, dengue viruses derived from the first- and eighth-plasmid passages achieved similar titers as the parent virus. They were also indistinguishable from the parent virus by the criteria of replication kinetics in mosquito and mammalian cell lines, and size and reactivity of selected viral proteins as detected with polyclonal and monoclonal antibodies. The cloning scheme and resultant recombinant plasmids based on high copy number cloning vectors allows greater flexibility in manipulation of dengue viral genome when compared with previous attempts employing low-copy number counterparts.

Cell Line↗

PubMed1986

Delimitation and characterization of cis-acting DNA sequences required for the regulated expression and transcriptional control of the chicken skeletal alpha-actin gene.

We have previously observed that DNA sequences within the 5'-flanking region of the chicken skeletal alpha-actin gene harbor a cis-acting regulatory element that influences cell type and developmental stage-specific expression (J. M. Grichnik, D. J. Bergsma, and R. J. Schwartz, Nucleic Acids Res 14:1683-1701, 1986). In this report we have constructed unidirectional 5'-deletion and region-specific deletion-insertion mutations of the chicken skeletal alpha-actin upstream region and inserted these into the chloramphenicol acetyltransferase expression vector pSV0CAT. These constructions were used to locate DNA sequences that are required for developmental modulation of expression when transfected into differentiating myoblasts. With this assay we have delimited the 5' boundary of a cis-acting regulatory element to ca. 200 base pairs upstream of the mRNA cap site. In addition, we have preliminarily identified DNA sequences that may be important subcomponents within this element. A second major focus of this study was to identify those DNA signals within the regulatory element that control transcription. Toward this end, the expression phenotypes of progressive 5'-deletion and deletion-insertion mutants of the 5'-flanking region of the chicken skeletal alpha-actin gene were assayed in microinjected Xenopus laevis oocytes. These experiments defined a cis-acting transcriptional control region having a 5' border 107 base pairs preceding the alpha-actin RNA cap site. Proximal and distal functionally important regions of DNA were identified within this element. These DNA signals included within their DNA sequences the "CCAAT" and "TATA" box homologies.

Actins↗

PubMed1985

Generation of glucocorticoid-responsive Moloney murine leukemia virus by insertion of regulatory sequences from murine mammary tumor virus into the long terminal repeat.

The glucocorticoid-regulatory sequences from the murine mammary tumor virus long terminal repeat (MMTV LTR) were introduced into the LTR of Moloney murine leukemia virus (M-MuLV) by recombinant DNA techniques. The site of insertion was in the M-MuLV LTR U3 region at -150 base pairs with respect to the RNA cap site. Infectious M-MuLVs carrying the altered LTRs (Mo + MMTV M-MuLVs) were recovered by transfection of proviral clones into NIH-3T3 cells. The Mo + MMTV M-MuLVs were hormonally responsive in that infection was 3 logs more efficient when performed in the presence of dexamethasone, irrespective of the orientation of the inserted MMTV sequences. However, even in the presence of hormone, the Mo + MMTV M-MuLVs were less infectious than wild-type M-MuLV. In contrast to the large effect on infectivity, dexamethasone induced virus-specific RNA levels in chronically Mo + MMTV M-MuLV-infected cells only two- to fourfold. Fusion plasmids between the altered LTRs and the bacterial chloramphenicol acetyltransferase gene allowed the investigation of LTR promoter strength by the transient chloramphenicol acetyltransferase expression assay. The chloramphenicol acetyltransferase assays indicated that the insertion of MMTV sequences into the M-MuLV LTR reduced promoter activity in the absence of glucocorticoids but that promoter activity could be induced two- to fivefold by dexamethasone. The Mo + MMTV M-MuLVs were also tested for the possibility that viral DNA synthesis or integration during initial infection was enhanced by dexamethasone. However, no significant difference was detected between cultures infected in the presence or absence of hormone. The insertion of MMTV sequences into an M-MuLV LTR deleted of its enhancer sequences did not yield infectious virus or active promoters, even in the presence of dexamethasone.

DNA, Recombinant↗

PubMed1986

Chicken U2 and U1 RNA genes are found in very different genomic environments but have similar promoter structures.

We have cloned and analyzed a gene that codes for chicken U2 small nuclear RNA (snRNA). In the haploid chicken genome, there are approximately 35-40 copies of the U2 RNA gene arranged in tandemly repeated units 5.35 kilobase pairs in length. This U2 gene organization contrasts with that of chicken U1 RNA genes, which are found in heterogeneous genomic environments. Although U snRNA genes are transcribed by RNA polymerase II, they lack the usual TATA and CAAT homologies found in the 5' control regions of most RNA polymerase II transcription units. Nevertheless, a comparison of chicken U2 and U1 RNA gene 5'-flanking DNA sequences reveals two upstream blocks of homology which are also evolutionarily conserved in U2 and U1 RNA genes of other vertebrate species. The first block of conserved sequence is centered around position -55 relative to the RNA cap site, and the other is located near position -200. Interestingly, stretches of sequence with the potential to form Z DNA are located either within or immediately adjacent to both of these two conserved upstream sequence elements, suggesting a possible role for Z DNA in U1/U2 gene expression. Moreover, the chicken U2 and U1 gene promoter regions also contain specific short sequences (i.e., the hexamer GGGCGG and the octamer ATGCAAAT) that have been shown to be required for the expression of a number of mRNA-encoding genes. These findings suggest that the transcription of snRNA genes is controlled by a complex set of factors, some shared with other RNA polymerase II transcription units and others which may be unique to the snRNA genes.

Animals↗