Template Activity Research Articles

Analysisin Vivoof Turnip Crinkle Virus Satellite RNA C Variants with Mutations in the 3′-Terminal Minus-Strand Promoter

Turnip crinkle virus and its associated RNA, sat-RNA C, share similar, but not identical hairpins near their 3′ ends and terminate with CCUGCCC-OH, which forms a single-stranded tail. With anin vitrotranscription system containing partially purified TCV RdRp, the 3′-terminal 29 bases making up the hairpin and single-stranded tail were previously demonstrated to be required for transcription, and alterations in the stem, but not the loop, could affect template activity (C. Song and A. E. Simon, 1995,J. Mol. Biol.254, 6–14). We have now analyzed sat-RNA C mutants in the 3′ hairpin for ability to accumulatein vivo.While active templatesin vitrowere able to accumulatein vivo,some very weak templatesin vitrowere also able to accumulatein vivowithout reversion or second-site alterations. Computer models of hairpin structure indicated that biologically active promoters could have hairpins less stable than wild type, with loops of variable length and sequence, and without a need for a 6-base single-stranded tail. In addition, transcripts containing compensatory exchanges in the upper stem region that had limited activityin vitrowere biologically activein vivo,indicating that positioning of specific bases in the stem is not required to produce an active minus-strand promoter.

Turnip Yellow Mosaic Virus RNA-Dependent RNA Polymerase: Initiation of Minus Strand Synthesisin Vitro

An RNA-dependent RNA polymerase (RdRp) activity was detergent-solubilized from the chloroplast membranes of Chinese cabbage leaves infected with turnip yellow mosaic virus (TYMV). The template-dependent, micrococcal nuclease-treated activity synthesized full-length minus strands from TYMV RNA and 3′-fragments as short as a 28-nucleotide-long RNA comprising the amino acid acceptor stem of the 3′-tRNA-like structure (TLS). Minus strands were shown to arise byde novoinitiation with the insertion of GTP opposite the penultimate (C) residue of the 3′-terminal -CCA. The TYMV RdRp activity was template specific in that poly(A) RNA was not copied, and alfalfa mosaic virus (AlMV) RNA, which does not contain a 3′-TLS, was a very poor template. However, other viral RNAs with a 3′-TLS andin vitrotranscripts of tRNAs were copied to varying degrees. Fully modified tRNAs were either inactive or poorly active templates, and AlMV 3′-RNA, even when provided with a 3′-terminal -ACCA, was not copied detectably. A potential role of the acceptor stem pseudoknot as a promoter element was assessed with mutations that drastically altered the structure and sequence of the pseudoknot, revealing only a twofold effect in decreasing template activity. The data show that RNAs with both a tRNA-like conformation and a -CCA 3′-terminus are potential templates for TYMV RdRp and suggest that promoter elements are not limited to the acceptor stem pseudoknot.

Recognition of bacteriophage Qβ plus strand RNA as a template by Qβ replicase: role of RNA interactions mediated by ribosomal proteins S1 and host factor

RNA-protein interactions between bacteriophage Qβ plus strand RNA and the components of the Qβ replicase system were studied by deletion analysis. Internal, 5′-terminal and 3′-terminal deletions were assayed for template activity with replicase in vitro. Of the two internal binding sites previously described for replicase, we found that the S-site (map position 1247 to 1346) could be deleted without any significant effect on template activity, whereas deletion of the M-site (map position 2545 to 2867) resulted in a strong inactivation and a high salt sensitivity of the residual activity. Binding complexes of the deletion mutant RNAs with the different proteins involved in Qβ RNA replication were analysed by electron microscopy. The formation of looped complex structures, previously reported and explained as simultaneous interactions with replicase at the S and the M-site, was abolished by deleting the S-site but, surprisingly, not by deleting the M-site. The same types of complexes observed with replicase were also formed with purified protein S1 (the α subunit of replicase), suggesting that these internal interactions with Qβ RNA are mediated by the S1 protein. The Qβ host factor, a protein required for the template activity of the Qβ plus strand, was reported earlier to form similar complexes by binding to the S and M-sites (or adjacent sites) and in addition to the 3′-end, resulting in double-looped structures. The patterns of looped complexes observed with the deletion mutant RNAs suggest that the binding of host factor might not involve the S and M-sites themselves but adjacent downstream sites. An additional internal host factor interaction near map position 2300 was detected with several mutant RNAs. Qβ RNA molecules with 3′-truncations formed 3′-terminal loops with similar efficiency as wild-type RNA, indicating that recognition of the 3′-end by host factor is not dependent on a specific 3′-terminal base sequence.

Transcriptional template activity of covalently modified DNA

The transcriptional template activity of covalent modified DNA is compared. 8-Methoxypsoralen (MOP), 3,4′dimethyl-8-methoxypsoralen (DMMOP) and benzopsoralen (BP) forming with DNA covalent complexes upon UV irradiation and exhibiting preference to pyrimidines, mostly thymines, differ in their cross-linking potency. MOP and DMMOP form both monoadducts and diadducts while no cross-links are formed by BP. Nitracrine (NC) forms covalent complexes with DNA upon reductive activation with dithiothreitol exhibiting a preference to purines and low cross-linking potency. Semilogarithmic plots of the relative template activity against the number of the drugs molecules covalently bound per 10 3 DNA nucleotides fit to regression lines corresponding to one-hit inactivation characteristics. The number of drug molecules decreasing RNA synthesis to 37% differ from 0.25 to 1.26 depending on the template used and the base preference but no dependence on the cross-linking potency was found.

Analysis of the 3′ Terminal Sequence Recognized by the Rift Valley Fever Virus Transcription Complex in Its Ambisense S Segment

A reconstituted transcription system composed of the Rift Valley fever phlebovirus (Bunyaviridae family) proteins L and N expressed via recombinant vaccinia viruses and an S-like model RNA containing the CAT gene in the antisense orientation, has been described previously by Lopezet al.(J. Virol.,1995, 69, 3972–3979). We extended the use of thisin vivosystem to determine the sequence at the 3′ end of the ambisense S segment recognized by the transcription complex. A mutational analysis of the sequences at the 3′ end of the S-like genomic or antigenomic RNA was undertaken. The data indicated that the minimal sequence required for transcription resides in the 13 first 3′ nucleotides of the genomic or antigenomic RNA. In these sequences, two regions appeared crucial: the bases at positions 3 to 8 and the purine at position 13. In addition, the terminal repeat ...GU could be deleted without affecting significantly the template activity of the RNA. These data support the prime and realign mechanism proposed recently for Bunya- and Arenaviruses

Helical structure formation between complementary oligonucleotides. Minimum chain length required for the template-directed synthesis of oligonucleotides.

Helix formation between various combinations of 3'-5' linked oligoribouridylates and oligoriboadenylates from dimer to dodecamer has been studied to gain information on the chain-length requirement for the template-directed condensation of oligoribonucleotides. We have measured the helix formation under high oligoribonucleotide concentration in the presence of magnesium ion at 0-50 degrees C by UV or CD, as many model processes of oligoribonucleotides replication have been carried out under such conditions. Adenylic acid, (pA), diadenylic acid, (pA)2, or triadenylic acid, (pA)3, forms a helix with poly(U) or oligo(U) with a chain length of more than eight. On the other hand, neither uridylic acid, (pU), nor diuridylic acid. (pU)2, can form a helix with oligo(A) or poly(A). Triuridylic acid, (pU)3, or the longer oligo(U) forms a helix with oligo(A) with a chain length of over six. The results suggest that a trimer is the minimum unit as an incorporating nucleotide for conducting any set of nonenzymatic template-directed synthesis, A --> U and U --> A, as the nonenzymatic template-directed condensation of oligoribonucleotides correlates well with the results of helix formation of complementary oligoribonucleotides. We have further found the partial helix formation between 2'-5' linked decauridylate, (pU)10, and pA or 2'-5' linked (pA)2 at 0 degrees C, which indicates the possibility of the template activity of long 2'-5' linked oligonucleotides for the nonenzymatic oligonucleotide synthesis.

P53 Inhibits RNA Polymerase III-Directed Transcription in a Promoter-Dependent Manner

Wild-type p53 represses Alu template activity in vitro and in vivo. However, upstream activating sequence elements from both the 7SL RNA gene and an Alu source gene relieve p53-mediated repression. p53 also represses the template activity of the U6 RNA gene both in vitro and in vivo but has no effect on in vitro transcription of genes encoding 5S RNA, 7SL RNA, adenovirus VAI RNA, and tRNA. The N-terminal activation domain of p53, which binds TATA-binding protein (TBP), is sufficient for repressing Alu transcription in vitro, and mutation of positions 22 and 23 in this region impairs p53-mediated repression of an Alu template both in vitro and in vivo. p53's N-terminal domain binds TFIIIB, presumably through its known interaction with TBP, and mutation of positions 22 and 23 interferes with TFIIIB binding. These results extend p53's transcriptional role to RNA polymerase III-directed templates and identify an additional level of Alu transcriptional regulation.

RNA-dependent RNA polymerase activity related to the 20S RNA replicon ofSaccharomyces cerevisiae

Saccharomyces cerevisiae contains two double-stranded RNA (dsRNA) viruses (L-A and L-BC) and two different single-stranded (ssRNA) replicons (20S RNA and 23S RNA). Replicase (dsRNA synthesis on a ssRNA template) and transcriptase (ssRNA synthesis on a dsRNA template) activities have been described for L-A and L-BC viruses, but not for 20S or 23S RNA. We report the characterization of a new in vitro RNA replicase activity in S. cerevisiae. This activity is detected after partial purification of a particulate fraction in CsCl gradients where it migrates at the density of free protein. The activity does not require the presence of L-A or L-BC viruses or 23S RNA, and its presence or absence is correlated with the presence or absence of the 20S RNA replicon. Strains lacking both this RNA polymerase activity and 20S RNA acquire this activity when they acquire 20S RNA by cytoduction (cytoplasmic mixing). This polymerase activity converts added ssRNA to dsRNA by synthesis of the complementary strand, but has no specificity for the 3' end or internal template sequence. Although it replicates all tested RNA templates, it has a template size requirement, being unable to replicate templates larger than 1 kb. The replicase makes dsRNA from a ssRNA template, but many single-stranded products due to a terminal transferase activity are also formed. These results suggest that, in contrast to the L-A and L-BC RNA polymerases, dissociation of 20S RNA polymerase from its RNA (or perhaps some cellular factor) makes the enzyme change its specificity.

RNA-dependent RNA polymerase activity related to the 20S RNA replicon of Saccharomyces cerevisiae.

Saccharomyces cerevisiae contains two double-stranded RNA (dsRNA) viruses (L-A and L-BC) and two different single-stranded (ssRNA) replicons (20S RNA and 23S RNA). Replicase (dsRNA synthesis on a ssRNA template) and transcriptase (ssRNA synthesis on a dsRNA template) activities have been described for L-A and L-BC viruses, but not for 20S or 23S RNA. We report the characterization of a new in vitro RNA replicase activity in S. cerevisiae. This activity is detected after partial purification of a particulate fraction in CsCl gradients where it migrates at the density of free protein. The activity does not require the presence of L-A or L-BC viruses or 23S RNA, and its presence or absence is correlated with the presence or absence of the 20S RNA replicon. Strains lacking both this RNA polymerase activity and 20S RNA acquire this activity when they acquire 20S RNA by cytoduction (cytoplasmic mixing). This polymerase activity converts added ssRNA to dsRNA by synthesis of the complementary strand, but has no specificity for the 3' end or internal template sequence. Although it replicates all tested RNA templates, it has a template size requirement, being unable to replicate templates larger than 1 kb. The replicase makes dsRNA from a ssRNA template, but many single-stranded products due to a terminal transferase activity are also formed. These results suggest that, in contrast to the L-A and L-BC RNA polymerases, dissociation of 20S RNA polymerase from its RNA (or perhaps some cellular factor) makes the enzyme change its specificity.

Differential binding of cAMP-responsive-element (CRE)-binding protein-1 and activating transcription factor-2 to a CRE-like element in the human tissue-type plasminogen activator (t-PA) gene promoter correlates with opposite regulation of t-PA by phorbol ester in HT-1080 and HeLa cells.

The human tissue-type plasminogen activator gene (t-PA) is induced by the phorbol ester, phorbol 12-myristate 13-acetate (PMA), in HeLa cells. Previous studies in transfected HeLa cells identified two cis-acting regulatory elements within the t-PA gene promoter responsible for both constitutive and PMA-inducible expression. One element differs from the consensus cAMP response element (CRE) by a single nucleotide substitution (referred to in this report as t-PACRE) and another which bears similarity to the AP-2 recognition sequence. In HT-1080 fibrosarcoma cells, t-PA mRNA levels are expressed at higher constitutive levels and are suppressed by PMA. Nuclear run-on transcription experiments indicate that PMA-mediated suppression of t-PA in these cells is associated with a decrease in t-PA gene template activity. We designed experiments to determine whether nuclear t-PACRE or AP-2-like binding proteins were differentially expressed in HeLa and HT-1080 cells and, accordingly, if these could be correlated with the opposite effect of PMA on t-PA expression. Band shift analyses indicated that the migration profiles of HeLa and HT-1080 nuclear proteins interacting with the AP-2-like site were indistinguishable; however, those produced with the t-PACRE binding site were qualitatively and quantitatively distinct. The distribution of t-PACRE binding proteins in these cells was investigated in a supershift assay using specific antibodies against members of the fos/jun and CRE-binding protein (CREB)/activating transcription factor (ATF) families. In HT-1080 cells, CREB-1 was the most prominent t-PACRE-binding activity detected and was greatly increased in cells treated with PMA. In contrast, CREB-1 activity was absent in HeLa cells, but antibodies specific for ATF-2 produced a marked supershifted complex which was unaffected by PMA treatment. Since CREB-1 can repress transcription of other target genes (including c-jun) via association with identical cis-acting CRE-like sequences, we suggest that the mechanism for the transcriptional down-regulation of t-PA by PMA in HT-1080 cells requires CREB-1 binding to the t-PACRE while ATF-2, by associating with the same site, plays a role in PMA-mediated induction of t-PA in HeLa cells.

Flanking sequences of an Alu source stimulate transcription in vitro by interacting with sequence-specific transcription factors

An Alu source gene, called the EPL Alu, was previously isolated by a phylogenetic strategy. Sequences flanking the EPL Alu family member stimulate its RNA polymerase III (Pol III) template activity in vitro. One cis-acting element maps within a 40-nucleotide region immediately upstream to the EPL Alu. This same region contains an Ap1 site which, when mutated, abolishes the transcriptional stimulation provided by this region. The flanking sequence, as assayed by gel mobility shift, forms sequence-specific complexes with several nuclear factors including Ap1. These results demonstrate that an an ancestral Alu source sequence fortuitously acquired positive transcriptional control elements by insertion into the EPL locus, thereby providing biochemical evidence for a model which explains the selective amplification of Alu subfamilies.

Identification of the minimal replicase and the minimal promoter of (-)-strand synthesis, functional in rotavirus RNA replication in vitro.

An in vitro replication system supporting the initiation and synthesis of complete rotavirus (-)-strands on (+)-strand template RNA (Chen et al., J Virol 68: 7030, 1994) was used to examine several parameters related to rotavirus RNA replication. Coexpression of VP1/2/3 in all possible combinations from baculovirus vectors revealed: [i] Virus-like particles (VLPs) were formed only if VP2 was present, and [ii] VP1/2 and VP1/2/3 VLPs had replicase activity in the in vitro system whereas VP2/3 and VP2 VLPs did not. Thus, the minimal replicase is composed of VP1 and VP2 and replicase activity is associated with VP1. In vitro replication reactions, using T7 transcripts of porcine rotavirus OSU genome segment 9 as reporter template, were performed to map cis-acting elements that regulate replication. Internal deletions and terminal truncations of the reporter RNA localized a replication signal, conferring full template activity, to the 5'-terminal 27 nucleotides (nt 1-27) and the 3'-terminal 26 nucleotides (nt 1037-1062). Further analysis showed that a minimal promoter of (-)-strand synthesis was contained in the 3'-terminal 7 nucleotides (nt 1056-1062); the sequence conserved at the 3'-terminus of all rotavirus genes. Hybrid constructs with this promoter had minimal, but detectable, template activity. This result indicated that upstream sequences between nucleotides 1037-1055 positively regulate the activity of the minimal promoter.

The Decay of Bacterial Messenger RNA

Publisher Summary The many demonstrations that the Escherichia coli (E. coli ) rne gene product (RNase E) is involved in messenger RNA (mRNA) decay have given real impetus to the study of this unusual protein's properties and role. The recent attention given to the polyadenylylation of bacterial mRNAs and the discovery that polyadenylylation plays a role in the turnover of an E. coli plasmid-specific RNA show that bacterial mRNA decay has similarities to the eukaryotic process. Functional decay of a messenger refers to the inactivation of a messenger's template activity, whereas chemical decay refers to the degradation of the mRNA itself. Functional decay, as a process distinct from the cleavages that initiate degradation, has been demonstrated for only a few mRNAs. Specific messengers have half-lives that range from about 0.5 to 20 minutes. The rates of decay of many messengers are actively regulated, thus, affecting the yields of the proteins synthesized. Unique to bacteria, portions of polycistronic mRNAs may decay at different rates, independent of the size of the segment or its position in the transcript. Overall, the decay of mRNA in bacterial cells is a major metabolic function. The turnover of mRNA constitutes about half of the cells' RNA synthesis; the actual amount is related to their growth rate.

Requirement of a 3′-Terminal Stem-loop in in VitroTranscription by an RNA-dependent RNA Polymerase

Partially purified RNA-dependent RNA polymerase (RdRp) isolated from plants infected with turnip crinkle virus (TCV) is capable of template- dependent synthesis of TCV-associated RNAs. To determine the cis-sequences required for the synthesis of TCV satellite (sat-) RNA C (−) strands in vitro, templates containing interior deletions were subjected to transcription using RdRp-active fractions. Results indicated that the promoter for (−)-strand synthesis was contained within the 3′-terminal 29 bases of the (+)-strand. Structural probing by enzymatic digestion and chemical modification revealed the presence of a hairpin structure within this terminal region. Compensatory exchanges of four bases in the lower stem or alterations in the sequence and size of the loop region did not affect in vitrotranscription, implying that the primary sequence in the loop and lower part of the stem is not important for interaction with the viral RdRp. However, single mutations in the base of the stem or double mutations in the upper stem strongly reduced template activity in vitro, suggesting that the stability of the hairpin is an important functional consideration. Relocation of the 3′-terminal 37 bases containing this stem-loop to an inactive template RNA rendered the resultant hybrid RNA competent for in vitrotranscription by RdRp activity, suggesting that the promoter for (−)-strand synthesis in vitrois completely contained within the 3′-terminal region.

Role of LFB3 in Cell-specific cAMP Induction of the Urokinase-type Plasminogen Activator Gene

In previous work we suggested that a kidney-specific transcription factor LFB3 cooperates with cAMP-response element (CRE)-binding proteins within a cAMP regulatory unit comprised of three protein-binding domains and located 3.4 kilobase pairs upstream of the urokinase-type plasminogen activator (uPA) gene in LLC-PK1 cells (Menoud, P.-A., Matthies, R., Hofsteenge, J., and Nagamine, Y. (1993) Nucleic Acids Res. 21, 1845-1852). The two domains contain a CRE-like sequence, and the third domain is recognized by LFB3. The absolute requirement of LFB3 as well as the cooperation among the three domains for cAMP regulation were confirmed by transient transfection assays in F9 teratocarcinoma cells, in which the level of LFB3 was negligible. Suspecting a possible feedback regulation of LFB3 mRNA expression during cAMP-dependent uPA gene induction in LLC-PK1 cells, we measured LFB3 mRNA levels after cAMP treatment and found a strong reduction. This reduction was not due to a change in template activity of the LFB3 gene because run-on transcription showed no significant change in LFB3 gene transcription. RNA synthesis inhibitor-chase experiments indicated that the down-regulation was post-transcriptional. Interestingly, when the inhibitor was added at the same time as cAMP, the cAMP-induced decrease in LFB3 mRNA levels was abrogated, suggesting that ongoing RNA synthesis is required for the decrease. Similar effects on LFB3 mRNA metabolism were observed with all agents that induce uPA mRNA in LLC-PK1 cells, including 12-O-tetradecanoylphorbol-13-acetate, okadaic acid, colchicine, and cytochalasin. We discuss the significance of this regulation in uPA gene expression.

RNA transcription from immobilized DNA templates.

We describe an RNA transcription protocol based on the multiple reuse of solid-phase synthetic DNA templates. The templates are assembled onto streptavidin-coated agarose beads via a single 5'-terminal biotin located on the noncoding template strand. Transcription occurs in an aqueous buffered suspension containing solid-phase DNA, dissolved enzyme (T7 RNA polymerase), and nucleoside triphosphate substrates (NTPs). A direct comparison of solution and solid-phase templates under standard transcription conditions reveals similar initial reaction rates and overall yields. Immobilized templates store stably for periods of several months and are easily recovered by mild centrifugation. We demonstrate the successive reuse of these templates throughout 15 rounds of transcription. The templates remained active, although an incremental decay in transcription was observed beyond five rounds. Template activity was partially restored by supplementing the support-bound oligonucleotide with fresh coding-strand DNA. These findings indicate that multiple reuse of template is a viable strategy for reducing the amount of DNA template required in RNA transcription.

Template Activity of Oligoribonucleotides Synthesized by the Phosphoramidite Method Using 2'-O-Tetrahydrofuranyl and 2'-O-Tetrahydropyranyl Protecting Groups

Abstract Solid-phase synthesis and functional activity of oligoribonucleotides containing native and modified translation initiation region (TIR) of phage MS2 and fr RNA replicase gene have been investigated.

Матричная активность плазмидоподобных ДНК митохондрий кукурузы в отношении синтеза ДНК

Матричная активность плазмидоподобных ДНК митохондрий кукурузы в отношении синтеза ДНК

Design of artificial short-chained RNA species that are replicated by Q beta replicase.

Different RNA species that are replicated by Q beta replicase have related secondary structures: for both plus and minus strands, "leader" stem structures were found at their 5' termini, while their 3' termini were unpaired. Parallel structures in complementary strands rather than antiparallel ones require the occurrence of wobble pairs and other imperfections in the stem regions. To test whether the leader structures are required for replication, artificial RNA sequences were synthesized by transcription from synthetic oligodeoxynucleotides with T7 RNA polymerase and assayed for their ability to be replicated by Q beta replicase. A synthetic short RNA species known to be replicated was amplified, forming a stable quasi-species; i.e., its sequence was conserved during hundreds of replication rounds. A synthetic mutant of this sequence that stabilized the leader in one strand but favored a 3'-terminal stem in the other one led to the complete loss of template activity. When new RNA sequences with the described structural requirements were designed and synthesized, their template activity was too low to be directly measurable; however, incubation with replicase produced replicating RNA whose sequence was closely related to the synthesized RNA species. The most likely interpretation is that the designed sequences were in a low montainous region in the replication fitness landscape and were optimized during amplification by Q beta replicase to a nearby fitness peak. The structural features postulated to be required for replication were not only conserved but even improved in the outgrowing mutants.(ABSTRACT TRUNCATED AT 250 WORDS)

The rat vault RNA gene contains a unique RNA polymerase III promoter composed of both external and internal elements that function synergistically.

A novel gene transcribed by RNA polymerase (pol) III has been recently identified that produces an RNA component of a large cytoplasmic ribonucleoprotein complex (Kickhoefer, V. A., Searles, R. P., Kedersha, N. L., Garber, M. E., Johnson, D. L., and Rome, L. H. (1993) J. Biol. Chem. 268, 7868-7873). Since sequence analysis revealed that this gene contains promoter elements from two different classes of RNA pol III gene promoters, we examined the function of the 5'-flanking type-3 and internal type-2 sequences on transcription activity and the production of stable transcription complexes. We find that the vRNA gene contains a novel RNA pol III promoter, where both the external and internal sequences are essential for template activity and for the productive interaction of TFIIIC with the internal elements. Thus, the vRNA gene represents the first example of a template that requires both type-2 and type-3 promoter elements that appear to function synergistically in the formation of productive transcription complexes. We have further examined the function of the unique arrangement of an internal A box and two B box elements. We find that at least one B element is required for template activity. In the absence of the 5'-flanking sequence the presence of both B elements inhibits transcription and the binding of TFIIIC. The formation of active complexes is restored when either the B2 element is inactivated or the distance separating the two B elements is increased. Therefore, the B2 element appears to negatively regulate template activity in the absence of the upstream sequences. This unique RNA pol III promoter arrangement may provide a novel mechanism for the regulation of vRNA gene activity.