Showing papers on "RNA-dependent RNA polymerase published in 1980"
TL;DR: This chapter describes the analysis of transcription maps of polyoma virus-specific RNA by 2-D nuclease S1 gel mapping and the standard procedures used for the preparation of viral nucleic acids and hybridization probes.
Abstract: Publisher Summary This chapter describes the analysis of transcription maps of polyoma virus-specific RNA by 2-D nuclease S1 gel mapping. The chapter describes the methodology used to map polyoma virus transcripts on the physical map of the viral genome. The ultimate characterization of viral RNA is the determination of the complete nucleotide sequence of individual RNA species. A rapid procedure for the analysis of labeled viral RNA using an adaptation of the Southern transfer technique (mini-blot hybridization) has also been described. The hybrids formed between polyoma virus RNA and DNA are digested with single-strand-specific endonuclease S1 and the resulting duplexes or their component DNA strands are sized by gel electrophoresis. The 2-D variation of this basic procedure is a general diagonal assay for splicing within RNA molecules. The chapter describes the standard procedures used for the preparation of viral nucleic acids and hybridization probes.
1,136 citations
TL;DR: The addition of a deoxyribonucleotide to the cleaved RNA can be regarded as the first step of ColE1 DNA synthesis and once it has served as a primer, the RNA is eliminated from the product by RNase H.
Abstract: A plasmid that consists of an 812-base-pair segment containing the replication origin of plasmid ColE1 and of a 1240-base-pair segment containing a beta-lactamase gene has been constructed. The plasmid DNA has three principal sites where transcription is initiated in vitro. One is located in the ColE1 segment 555 nucleotides upstream from the origin. Most transcription from this site extends past the origin; some of the transcripts form hybrids spontaneously with the template at their 3' portions. Cleavage of these transcripts by RNase H generates 3' termini at the origin region. When DNA polymerase I is included in the reaction along with RNA polymerase and RNase H, dAMP or dCMP is added directly onto the cleaved RNA molecules, most of which retain the intact 5' terminus. The addition of a deoxyribonucleotide to the cleaved RNA can be regarded as the first step of ColE1 DNA synthesis. Once it has served as a primer, the RNA is eliminated from the product by RNase H.
440 citations
TL;DR: 5S ribosomal RNA specifically inhibits transcription of cloned repeating units of 5S DNA in a nuclear extract of Xenopus oocytes, indicating the presence of large amounts of this protein in these cells can account for both the high rate of synthesis and the subsequent storage of5S RNA to ribosome synthesis.
Abstract: 5S ribosomal RNA specifically inhibits transcription of cloned repeating units of 5S DNA in a nuclear extract of Xenopus oocytes. The inhibition can be explained by the interaction of 5S RNA with a transcription factor that binds specifically to a control region located within the 5S RNA gene. This transcription factor is identical to an abundant cytoplasmic protein that is known to be complexed with 5S RNA in immature Xenopus oocytes. Thus the presence of large amounts of this protein in these cells can account for both the high rate of synthesis and the subsequent storage of 5S RNA to ribosome synthesis.
421 citations
TL;DR: Specific contacts between the Escherichia coli RNA polymerase and the phosphates and purine bases of the A3 promoter of phage T7 cluster into three regions located approximately 10, 16, and 35 base pairs before RNA initiation site.
Abstract: Specific contacts between the Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyl-transferase, EC2.7.7.6) and the phosphates and purine bases of the A3 promoter of phage T7 cluster into three regions located approximately 10, 16, and 35 base pairs before RNA initiation site. Two of these contain nucleotide sequences that are fairly conserved among many promoters, known as the "Pribnow box" and "-35 region" homologies; the third, just upstream from the Pribnow box, is not conserved. The polymerase binds preferentially to the coding strand and for the most part touches only one face of the DNA helix.
391 citations
TL;DR: The similarity of the morphology and of DNA composition, the homology of the component patterns of DNA-dependent RNA polymerases and their immunochemical crossreactivity support the conclusion that several extreme thermoacidophiles are related to each other.
Abstract: The similarity of the morphology and of DNA composition, the homology of the component patterns of DNA-dependent RNA polymerases and their immunochemical crossreactivity support the conclusion that several extreme thermoacidophiles are related to each other. We name two new species of the genus Sulfolobus. The first, Sulfolobus solfataricus (DSM 1616 and DSM 1617) has the same GC content in its DNA and the same general properties as S. acidocaldarius, but differs significantly from the latter species in the molecular weights of the 11 components of its RNA polymerase and in the salt requirements of this enzyme. The second, Sulfolobus brierleyi, DSM 1651, differs from S. acidocaldarius in several respects. The cells show much less stability at neutral pH. The GC content is significantly lower. The RNA polymerase lacks two components present in the enzymes from the other species. The residual 9 components show larger size differences from the homologous subunits of the S. acidocaldarius enzyme. Like the enzyme from S. solfataricus, the polymerase from S. brierleyi yields an incomplete immunochemical crossreaction with an antibody against the RNA polymerase from S. acidocaldarius. The isolates DSM 1616 and DSM 1617 of Sulfolobus solfataricus are probably identical with or similar to the “Caldariella” strains MT 3 and MT 4, isolated by de Rosa et al. (1975). Like all other known archaebacterial RNA polymerases the enzymes from these species are insensitive to rifampicin and streptolydigin.
386 citations
TL;DR: Fractionated the KB S-100 and have found that multiple components are essential for the accurate transcription of these genes.
308 citations
TL;DR: Messenger RNA synthesis by the DNA tumour viruses proceeds by a complex but versatile series of transcription and RNA processing steps which allow them to use their genetic information to maximum advantage.
Abstract: Messenger RNA synthesis by the DNA tumour viruses proceeds by a complex but versatile series of transcription and RNA processing steps. The major mechanistic features of this pathway are probably very similar to those used by the animal cell host itself. The viruses have, however, evolved intricate arrangements of protein coding sequences and sites for RNA initiation, polyadenylation and splicing which allow them to use their genetic information to maximum advantage.
186 citations
TL;DR: Evidence is presented that a homogeneous cytoplasmic species known as 7S RNA is the only abundant RNA in uninfected HeLa cells which can form strong hybrids with the dominant family of middle repetitive DNA sequences in the human genome.
170 citations
TL;DR: The viral polypeptide, p63, is an RNA-dependent RNA polymerase that can copy poliovirion RNA when oligouridylic acid is used as a primer.
Abstract: A poliovirus-specific RNA-dependent RNA polymerase was isolated from a cytoplasmic extract of infected HeLa cells and was shown to copurify with a single virus-specific protein. The polymerase was isolated from cells labeled with [35S]-methionine and was fractionated from other soluble cytoplasmic proteins by ammonium sulfate precipitation, phosphocellulose chromatography, gel filtration on Sephacryl S-200, and chromatography on hydroxylapatite. The activity of the enzyme was measured by using either polyadenylic acid or poliovirion RNA as a template in the presence of an oligouridylic acid primer. A single virus-specific protein that had an apparent molecular weight of 63,000 (p63) was found to copurify with this activity. Host-coded proteins were present in reduced molar amounts relative to p63. Noncapsid viral protein 2 (NCVP2) and other viral proteins were clearly separated from p63 by gel filtration on Sephacryl S-200. Polymerase activity coeluted from the column precisely with p63. NCVP2 was totally inactive as an RNA polymerase and did not stimulate the polymerase activity of p63. The purified enzyme sedimented at about 4S on a glycerol gradient and thus appeared to be a monomer of p63. Two-dimensional gel electrophoresis of the polymerase protein indicated that it had an isoelectric point of about 7.5. Thus, the viral polypeptide, p63, as defined by the above physical parameters, is an RNA-dependent RNA polymerase that can copy poliovirion RNA when oligouridylic acid is used as a primer.
154 citations
TL;DR: Physical association of the three enzymatic activities appears to be an efficient mechanism for carrying out the following successive steps in cap formation: removal of the gamma-phosphate preceded capping.
151 citations
TL;DR: Three of the viral systems may well have shared genes but the double-stranded RNA viruses appear to represent a very different evolutionary line.
Abstract: These arguments lead to the suggestion that four independent evolutionary lines exist within the general group of RNA viruses. These are positive strand viruses, negative strand viruses, double stranded viruses, and retroviruses. Three of the viral systems may well have shared genes but the double-stranded RNA viruses appear to represent a very different evolutionary line.
TL;DR: The results indicate that the cap 1 structure found in all mammalian cellular mRNAs is more stringently required for priming influenza virus RNA transcription than for translation in cell-free systems.
Abstract: The ability of eukaryotic mRNAs to serve as primers for influenza virus RNA transcription depends on the presence of a 5'-terminal methylated can structure, the absence of which eliminates essentially all priming activity [Plotch, S. J., Bouloy, M. & Krug, R. M. (1979) Proc. Natl. Acad. Sci. USA 76, 1618-1622]. The present study was undertaken to determine the extent to which each of the methyl groups in the cap influences the priming activity of a mRNA. To assess the importance of the 2'-O-methyl group on the penultimate base of the cap, we used several plant viral RNAs containing the monomethylated cap 0 structure, m7GpppG. Brome mosaic virus (BMV) RNA 4 stimulated influenza virus RNA transcription only about 10-15% as effectively as did globin mRNA, which has a cap with a 2'-O-methyl group. When the cap of BMV RNA 4 was enzymatically 2'-O-methylated, its priming activity was increased 14-fold. Qualitatively similar results were obtained with other plant virus RNAs. To assess the importance of the terminal 7-methyl group, BMV RNA 4 containing the cap structure GpppGm was prepared by a series of chemical and enzymatic steps. These molecules were found to be only about 15% as active in priming as BMV RNA 4 molecules containing the fully methylated cap, m7GpppGm, indicating that the terminal 7-methyl group also strongly enhances priming activity. These results indicate that the cap 1 structure (m7GpppXm) found in all mammalian cellular mRNAs is more stringently required for priming influenza virus RNA transcription than for translation in cell-free systems.
TL;DR: A reconstitution experiment in which s Sigma 37 was added to core RNA polymerase strongly suggests that sigma 37 is responsible for the transcriptional specificity of the modified RNA polymerases.
Abstract: A modified form of Bacillus subtilis RNA polymerase (RNA nucleotidyltransferase) has been isolated that exhibits distinctive transcriptional specificity. This modified enzyme transcribes two cloned genes from the purA-cysA region of the B. subtilis chromosome whose expression in vivo is associated with the process of sporulation. Neither of these genes is transcribed by the usual form of B. subtilis RNA polymerase holoenzyme containing a sigma factor of 55,000 daltons (sigma 55). The modified RNA polymerase lacks sigma 55 but contains a newly identified subunit of 37,000 daltons termed sigma 37. A reconstitution experiment in which sigma 37 was added to core RNA polymerase strongly suggests that sigma 37 is responsible for the transcriptional specificity of the modified RNA polymerase. Sigma 37 apparently acts at the level of promoter recognition; this transcriptional determinant enabled core RNA polymerase to form stable binary and ternary ("initiation") complexes with endonuclease restriction fragments containing promoters for the cloned B. subtilis genes.
TL;DR: The interaction between rho protein, an RNA synthesis termination factor from Escherichia coli, and synthetic RNA polymers has been studied using a membrane filter binding technique and by the inhibition of enzymes that digest RNA to suggest that rho forms a very stable, salt-resistant complex with poly(C).
TL;DR: With the purified replicase-host factor combination, only poly(A)-containing RNAs were copied, and a preference for poliovirus RNA was shown, suggesting that the factor acts at the initiation step of RNA replication.
TL;DR: It is concluded that the minus-strand polymerase regulates the rate of SFV plus- Strand RNA synthesis by determining the number of minus-Strand templates and that the synthesis of the minus -strand templates is regulated at the level of translation by a mechanism which utilizes one or more short-lived polymerase proteins.
Abstract: Semliki Forest virus (SFV)-infected BHK-21, Vero, and HeLa cells incorporated [3H]uridine into 42S and 26S plus-strand RNA and into viral minus-strand RNA (complementary to the 42S virion RNA) early in the infectious cycle. Between 3 and 4 h postinfection, the synthesis of minus-strand RNA ceased in these cultures, although the synthesis of plus-strand RNA continued at a maximal rate. At the time of cessation of minus-strand RNA synthesis, two changes in the pattern of viral protein synthesis were detected: a decrease in the translation of nonstructural proteins and an increase in the translation of the viral structural proteins. Addition of cycloheximide and puromycin to cultures of SFV-infected BHK cells actively synthesizing both viral plus- and minus-strand RNA resulted within 15 to 30 min in the selective shutoff of minus-strand RNA synthesis. Removal of the cycloheximide-containing medium led to the resumption of minus-strand synthesis and to an increased rate of viral RNA synthesis. We conclude that the minus-strand polymerase regulates the rate of SFV plus-strand RNA synthesis by determining the number of minus-strand templates and that the synthesis of the minus-strand templates is regulated at the level of translation by a mechanism which utilizes one or more short-lived polymerase proteins. Images
TL;DR: Fingerprinting showed that the intracellular RNAs constitute a nested set such that the nucleotide sequence of each RNA is contained within all larger RNAs and each larger RNA contains an additional sequence congruent with its greater size.
Abstract: We examined the synthesis of intracellular RNA in primary chicken embryo kidney cells infected with the avian coronavirus infectious bronchitis virus. Infected cells were labeled with (32)P(i) in the presence of actinomycin D for the duration of the viral multiplication cycle, and nucleic acids were extracted, denatured, and analyzed on agarose slab gels. Six major RNA species were found. None of these RNAs was found in extracts of mock-infected cells. All six of the virus-specified RNAs (designated species A through F) were single stranded, and RNA species F had the same electrophoretic mobility as purified viral genome RNA. The molecular weights of the five subgenomic RNAs were estimated to be 0.8 x 10(6), 0.9 x 10(6), 1.3 x 10(6), 1.5 x 10(6), and 2.6 x 10(6) for species A through E, respectively. All of the RNAs were polyadenylated and are therefore likely to be viral mRNA's. The RNAs were synthesized in approximately constant proportions throughout the viral multiplication cycle. Intracellular RNA species A, B, C, D, and F and the purified viral genome were analyzed by RNase T(1) fingerprinting. The results confirmed the identification of RNA species F as the intracellular genome and the derivation of the four smaller RNAs from the genome. Fingerprinting also showed that the intracellular RNAs constitute a nested set such that the nucleotide sequence of each RNA is contained within all larger RNAs and each larger RNA contains an additional sequence congruent with its greater size. Finally, the possible modes of transcription and translation of the infectious bronchitis virus RNAs are discussed.
TL;DR: A block in the transition from immediate-early to early RNA did not account for the extended period of time that elapses between the time of infection and the initiation of viral DNA synthesis, and the number of cells that synthesized detectable amounts of viral antigens increased steadily during this time.
Abstract: The rate of accumulation of cytomegalovirus transcripts in permissively infected human embryonic lung (HEL) cells was analyzed at various times after infection by hybridization of infected cell RNA to undigested or restriction endonuclease-digested cytomegalovirus DNA fixed to nitrocellulose filters. Differences in patterns of transcript accumulation were determined by measuring the abundance levels of RNA which hybridized to different HindIII-, XbaI-, or EcoRI-generated fragments of cytomegalovirus DNA. The results showed that a small but significant amount of cytomegalovirus RNA was detectable within the first 3 h after infection and that the rate of accumulation of these transcripts was static during the first 24 h, but increased thereafter. In general, the viral transcripts accumulating in infected cells could be divided into three classes. Immediate-early RNA (synthesized in the absence of protein synthesis in infected cells) hybridizes predominantly to a very restricted part of the genome and can be identified during the first 2 to 4 h postinfection. Early RNA (synthesized up to about 24 h after infection) originates from most regions of the genome but is characterized by the presence of transcripts which hybridize in great abundance to certain fragments. Late RNA (synthesized after 24 h, i.e., after the onset of viral DNA synthesis) hybridizes in approximately equal abundance to most regions of the viral genome. These results showed that a block in the transition from immediate-early to early RNA did not account for the extended period of time that elapses between the time of infection and the initiation of viral DNA synthesis. Interestingly, despite rapid adsorption and penetration and a static level of accumulation of transcripts in the cultures during the first 24 h, the number of cells that synthesized detectable amounts of viral antigens increased steadily during this time.
TL;DR: The absence of antibodies directed against proteins possibly contaminating the antigens used for immunization allowed us to identify RNA polymerase unequivocally in interbands and puffs of polytene chromosomes.
Abstract: Monoclonal antibodies were raised against purified RNA polymerase II ( or B) from Drosophila melanogaster. The antibody produced by one hybridoma cell clone was found to be directed against the two large subunits of the enzyme. The absence of antibodies directed against proteins possibly contaminating the antigens used for immunization allowed us to identify RNA polymerase unequivocally in interbands and puffs of polytene chromosomes. Within a single heat shock puff (87C1) RNA polymerase was found to be clustered in two separate areas suggesting two distinct regions of RNA polymerase activity in this puff.
TL;DR: The results indicate that viral mRNA synthesis occurs in vitro by multiple initiations at different promoter sites on the genome RNA, and that the elongation and completion of the individual mRNAs depend on prior transcription of 3' proximal genes.
TL;DR: Analysis of RNA encapsidation and infectivity assays of protoplast homogenates revealed that virion formation was greatest between 10 and 25 h after inoculation, and double-stranded replicative forms of the four virus RNAs were observed.
Abstract: Summary
The rates of virus RNA synthesis and virion accumulation were investigated in brome mosaic virus-infected barley protoplasts. Single-stranded virus RNAs could be detected as early as 6 h after inoculation. Only RNA components 1 and 2 were detected at this time, suggesting that their synthesis is initiated relatively early in infection. The RNAs were synthesized at similar rates from 16 to 35 h post inoculation with maximal synthesis until approx. 25 h after inoculation. Double-stranded replicative forms of the four virus RNAs were observed. Their synthesis was first detectable at 6 h post inoculation and followed a time course similar to that of the single-stranded RNA species. Analysis of RNA encapsidation and infectivity assays of protoplast homogenates revealed that virion formation was greatest between 10 and 25 h after inoculation. All four RNAs were present in virions at 10 h post inoculation. Particles containing RNA 3 and RNA 4 accumulated at a faster rate than particles containing RNA 1 or RNA 2.
TL;DR: This chapter discusses the RNA polymerase nascent product analysis and the primed RNA synthesis method, which may be effective for sequence analysis of DNA segments containing heterogeneous ends and no substrate sites of known restriction endonucleases.
Abstract: Publisher Summary This chapter discusses the RNA polymerase nascent product analysis. Synthesis of RNA with RNA polymerase is initiated with purine nucleoside triphosphates. Under a reduced NTP concentration, RNA synthesis can be initiated with oligonucleotides as primer at defined sites of single-stranded DNA. By applying this principle, the entire region of a DNA segment is able to be transcribed into RNA sequences from which the sequence of the template is deduced. This procedure is applied for the sequence determination of the lac operator and of fd promoters. As new methods for direct DNA sequencing were developed, the primed RNA synthesis method is used because of complexity of the sequencing procedure. This method may be effective for sequence analysis of DNA segments containing heterogeneous ends and no substrate sites of known restriction endonucleases. The new sequencing procedure that resolves partial nuclease digests of terminally labeled RNA by gel electrophoresis would be more useful because oligonucleotides with 5'-OH ends can be used as primer, and RNA chains primed with such oligonucleotides are selectively labeled with 32 P in the polynucleotide kinase reaction.
TL;DR: The vaccinia polymerase is capable of transcribing through an RNA:DNA hybrid with concomitant displacement of the RNA strand, suggesting that the polymerase binds to and transcribes short single-stranded regions present in superhelical DNAs.
TL;DR: The reverse transcriptase, and not the viral RNA, is probably the major factor determining which tRNA's are incorporated into avian sarcoma virus particles.
Abstract: Mutants of avian sarcoma virus which lack a functional DNA polymerase were found to be nonselective in the incorporation of host cell tRNA's into virus particles. In contrast, mutants which possess a functional DNA polymerase but lack the viral genome RNA contained a specific subset of the host cell tRNA population, indistinguishable from that of the wild-type virus. Thus the reverse transcriptase, and not the viral RNA, is probably the major factor determining which tRNA's are incorporated into avian sarcoma virus particles. Supporting evidence was obtained in an in vitro binding assay between purified reverse transcriptase and unfractionated cellular tRNA's. However, the subset of tRNA's which associated with the genome in the 70S complex was determined primarily by the viral RNA. In the absence of DNA polymerase, the 70S RNA complex in mature virus particles contained the normal complement of associated tRNA's with the exception of tRNATrp, the primer for RNA-directed DNA synthesis.
TL;DR: It is shown by RNase T1 oligonucleotide fingerprinting that RNA E is also a member of the nested set and discussed in relation to the multiplication strategy both of coronaviruses and of other RNA-containing viruses.
Abstract: Avian infectious bronchitis virus, a coronavirus, directed the synthesis of six major single-stranded polyadenylated RNA species in infected chicken embryo kidney cells. These RNAs include the intracellular form of the genome (RNA F) and five smaller RNA species (RNAs A, B, C, D, and E). Species A, B, C, and D are subgenomic RNAs and together with the genome form a nested sequence set, with the sequences of each RNA contained within every larger RNA species (D. F. Stern and S. I. T. Kennedy, J. Virol 34:665-674, 1980). In the present paper we show by RNase T1 oligonucleotide fingerprinting that RNA E is also a member of the nested set. Partial alkaline fragmentation of the genome followed by sucrose fractionation, oligodeoxythymidylate-cellulose chromatography, and RNase T1 fingerprinting gave a partial 3'-to-5' oligonucleotide spot order. A comparison of the oligonucleotides of each of the five subgenomic RNAs with this spot order established that all of the RNAs are comprised of nucleotide sequences inward from the 3' end of the genome. This result is discussed in relation to the multiplication strategy both of coronaviruses and of other RNA-containing viruses.
TL;DR: Virus-specific protein and RNA syntheses have been analyzed in chicken embryo fibroblast cells infected with two group IV temperature-sensitive mutants of influenza A (fowl plague) virus in which the ts lesion maps in RNA segment 8.
Abstract: Virus-specific protein and RNA syntheses have been analyzed in chicken embryo fibroblast cells infected with two group IV temperature-sensitive (ts) mutants of influenza A (fowl plague) virus in which the ts lesion maps in RNA segment 8 (J. W. Almond, D. McGeoch, and R. D. Barry, Virology 92:416-427, 1979), known to code to code for two nonstructural proteins, NS1 and NS2. Both mutants induced the synthesis of similar amounts of all the early virus-specific proteins (P1, P2, P3, NP, and NS1) at temperatures that were either permissive (34 degrees C) or nonpermissive (40.5 degrees C) for replication. However, the synthesis of M protein, which normally accumulates late in infection, was greatly reduced in ts mutant-infected cells at 40.5 degrees C compared to 34 degrees C. The NS2 protein was not detected at either temperature in cells infected with one mutant (mN3), and was detected only at the permissive temperature in cells infected with mutant ts47. There was no overall reduction in polyadenylated (A+) complementary RNA, which functions as mRNA, in cells infected with these mutants at 40.5 degrees C compared to 34 degrees C, nor was there any evidence of selective accumulation of this type of RNA within the nucleus at the nonpermissive temperature. No significant differences in ts mutant virion RNA transcriptase activity were detected by assays in vitro at 31 and 40.5 degrees C compared to wild-type virus. Virus-specific non-polyadenylated (A-) complementary RNA, which is believed to act as the template for new virion RNA production, accumulated normally in cells at both 34 and 40.5 degrees C, but at 40.5 degrees C accumulation of new virion RNA was reduced by greater than 90% when compared to accumulation at 34 degrees C.
TL;DR: In vitro RNA synthesis by ASF virus requires higher concentrations of ATP than of GTP or CTP, and the RNA product has a sedimentation rate of 6–14 S and anneals specifically with virus DNA.
TL;DR: The Saccharomyces cerevisiae mutant rpo B1 produces a DNA-dependent RNA polymerase B defective in RNA synthesis in vitro that alters the B220 subunit, suggesting a role for this subunit in RNA chain elongation and in the association of the B32 and B16.5 subunits to the RNA polymerases molecule.
TL;DR: The four major adeno-associated virus type 2 (AAV2)-specific RNAs were mapped on the linear viral genome by a variety of biochemical techniques, including S1 nuclease and exonuclease VII mapping, RNA gel-transfer hybridization, and analysis of reverse transcriptase extension products.
Abstract: The four major adeno-associated virus type 2 (AAV2)-specific RNAs were mapped on the linear viral genome by a variety of biochemical techniques, including S1 nuclease and exonuclease VII mapping, RNA gel-transfer hybridization, and analysis of reverse transcriptase extension products. All the major AAV2 RNAs were derived from the minus DNA strand and had 3' termini at position 96. The nucleus-specific 4.3- and 3.6-kilobase (kb) RNAs had 5' termini at positions 6 and 19, respectively. The 5' terminus of the 2.6-kb RNA mapped to position 38.5. The predominant 2.3-kb AAV2 mRNA was spliced and contained a short leader sequence (approximately 50 nucleotides) which mapped to position 38.5, coincident with the 5' terminus of the 2.6-kb RNA. The 5' end of the body of the 2.3-kb RNA mapped to position 46.5. These results are discussed in terms of the involvement of single versus multiple promoters (for transcription) and RNA splicing mechanisms in the generation of the AAV2 RNAs.
TL;DR: The SP01 promoter, which was isolated from B. subtilis phage SP01 DNA, is homologous to promoters for Escherichia coli RNA polymerase; the sequences of the “-35 region” and the "Pribnow box" were 5′TTGACT and 5′CATAAT, respectively.
Abstract: We report the nucleotide sequence of a promoter recognized by RNA polymerase from the gram-positive bacterium Bacillus subtilis. This promoter, which was isolated from B. subtilis phage SP01 DNA, is homologous to promoters for Escherichia coli RNA polymerase; the sequences of the "-35 region" and the "Pribnow box" were 5'TTGACT and 5'CATAAT, respectively (T is the thymine analog 5-hydroxymethyluracil in SP01 DNA). These sequences each differed by only a single base pair from the preferred sequences for E. coli promoters. Not surprisingly, the SP01 promoter was actively transcribed in vitro by E. coli RNA Polymerase as well as by B. subtilis RNA polymerase.