Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Direct RNA sequencing”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 901 records · Page 50Linked to original sources
PubMed1989

Mutation changing the specificity of an RNA polymerase sigma factor.

We describe a mutation that changes the fine specificity of promoter selection by a secondary form of RNA polymerase holoenzyme in Bacillus subtilis. The product of regulatory gene spo0H is an RNA polymerase sigma factor called sigma H, which directs transcription of a sporulation gene known as spoVG. We show that the spo0H mutation spo0H81, which blocks transcription from the wild-type spoVG promoter, enhances transcription from a mutant form of the spoVG promoter (spoVG249) bearing a severe down-mutation (a G.C to A.T transition) at position -13 in the "-10 region." Suppression of the spoVG249 mutation is specific in the sense that the transcription from several other spoVG mutant promoters was not restored by the mutant sigma. Evidently, spo0H81 is a change-of-specificity mutation that alters sigma H-RNA polymerase in a way that decreases its capacity to use the wild-type spoVG promoter, while increasing its capacity to use the mutant promoter. Transcription experiments in vitro using RNA polymerase containing the wild-type or mutant sigma support this interpretation. The spo0H81 mutation causes a threonine (Thr100) to isoleucine substitution in a region of sigma H that is highly homologous among sigma factors of diverse origins. We discuss the possibility that Thr100 is an amino acid-base-pair contact site and that sigma factors contact the -10 region of their cognate promoters by means of amino acid residues in this highly conserved region.

Bacillus subtilis↗
PubMed1994

Analysis of the expression and regulation of the gerB spore germination operon of Bacillus subtilis 168.

The gerB spore germination operon of Bacillus subtilis 168 is a homologue of the gerA spore germination operon. The expression and regulation of the gerB operon has been examined using a lacZ transcriptional fusion and the transcriptional start defined. The gerB operon is expressed during sporulation under the control of RNA polymerase containing the forespore-specific sigma factor, delta G. This is a further homology to the gerA operon, which is similarly regulated. It is predicted from the localization of expression and the encoded primary sequences that the GerB proteins are located at the inner spore membrane.

Bacillus subtilis↗
PubMed2000

Expression of functional influenza virus RNA polymerase in the methylotrophic yeast Pichia pastoris.

Influenza virus RNA polymerase with the subunit composition PB1-PB2-PA is a multifunctional enzyme with the activities of both synthesis and cleavage of RNA and is involved in both transcription and replication of the viral genome. In order to produce large amounts of the functional viral RNA polymerase sufficient for analysis of its structure-function relationships, the cDNAs for RNA segments 1, 2, and 3 of influenza virus A/PR/8, each under independent control of the alcohol oxidase gene promoter, were integrated into the chromosome of the methylotrophic yeast Pichia pastoris. Simultaneous expression of all three P proteins in the yeast P. pastoris was achieved by the addition of methanol. To purify the P protein complexes, a sequence coding for a histidine tag was added to the PB2 protein gene at its N terminus. Starting from the induced P. pastoris cell lysate, we partially purified a 3P complex by Ni(2+)-agarose affinity column chromatography. The 3P complex showed influenza virus model RNA-directed and ApG-primed RNA synthesis in vitro but was virtually inactive without addition of template or primer. The kinetic properties of model template-directed RNA synthesis and the requirements for template sequence were analyzed using the 3P complex. Furthermore, the 3P complex showed capped RNA-primed RNA synthesis. Thus, we conclude that functional influenza virus RNA polymerase with the catalytic properties of a transcriptase is formed in the methylotrophic yeast P. pastoris.

Base Sequence↗
PubMed1988

At least three different RNA polymerase holoenzymes direct transcription of the agarase gene (dagA) of Streptomyces coelicolor A3(2).

Using a combination of gel filtration and anion exchange FPLC, three different RNA polymerase holoenzymes from Streptomyces coelicolor A3(2) have been separated. Each holoenzyme transcribes from only one of the four promoters of the S. coelicolor A3(2) dagA gene. Holoenzyme reconstitution experiments identified the sigma factors responsible for recognition of two of the promoters. The previously identified E sigma 49 transcribes from the dagA p3 promoter, whereas a novel species, E sigma 28, recognizes the dagA p2 promoter. Circumstantial evidence suggests that the third holoenzyme, which transcribes from the dagA p4 promoter, is the previously identified E sigma 35. This level of transcriptional complexity supports the idea that RNA polymerase heterogeneity may play a central role in the regulation and coordination of gene expression in this biochemically and morphologically complex bacterium.

Base Sequence↗
PubMed1995

The sigma-70 subunit from Escherichia coli C differs from that of E. coli K-12.

The nucleotide sequence of the rpoD gene (encoding the primary sigma-70 (sigma 70) subunit of RNA polymerase) from Escherichia coli C was determined. This gene differs from that of E. coli K-12 by a 30-bp deletion and five single-bp substitutions, resulting in a sigma 70 subunit with three amino acid (aa) changes and a deletion of ten acidic aa.

Amino Acid Sequence↗
PubMed1990

Identification of a region of the bacteriophage T3 and T7 RNA polymerases that determines promoter specificity.

Bacteriophages T7 and T3 encode DNA-dependent RNA polymerases that are 82% homologous, yet exhibit a high degree of specificity for their own promoters. A region of the RNA polymerase gene (gene 1) that is responsible for this specificity has been localized using two approaches. First, the RNA polymerase genes of recombinant T7 x T3 phage that had been generated in other laboratories in studies of phage polymerase specificity were characterized by restriction enzyme mapping. This approach localized the region that determines promoter specificity to the 3' end of the polymerase gene, corresponding to the carboxyl end of the polymerase protein distal to amino acid 623. To define more closely the region of promoter specificity, a series of hybrid T7/T3 RNA polymerase genes was constructed by in vitro manipulation of the cloned genes. The specificity of the resulting hybrid RNA polymerases in vitro and in vivo indicates that an interval of the polymerase that spans amino acids 674 to 752 (the 674 to 752 interval) contains the primary determinant of promoter preference. Within this interval, the amino acid sequences of the T3 and T7 enzymes differ at only 11 out of 79 positions. It has been shown elsewhere that specific recognition of T3 and T7 promoters depends largely upon base-pairs in the region from -10 to -12. An analysis of the preference of the hybrid RNA polymerases for synthetic T7 promoter mutants indicates that the 674 to 752 interval is involved in identifying this region of the promoter, and suggests that another domain of the polymerase (which has not yet been identified) may be involved in identifying other positions where the two consensus promoter sequences differ (most notably at position -15).

Amino Acid Sequence↗
PubMed2006

Extensive RNA editing in transcripts from the PsbB operon and RpoA gene of plastids from the enigmatic moss Takakia lepidozioides.

RNA editing is a post-transcriptional process that changes individual nucleotides in transcripts, and usually occurs in the plastids of land plants. The number of RNA editing sites in a plastid is significantly divergent in bryophytes, ranging from zero in liverworts to almost 1,000 sites in hornworts. In this study, we identified 132 RNA editing sites in the transcripts of six genes from the psbB operon and the rpoA of the moss Takakia lepidozioides. This is the highest number of RNA editing sites known in this region among land plant species. All were cytidine-to-uridine conversions. More than 91% of RNA editing occurred at the first or second codon positions, and it altered amino acid identity. Six editing sites created new translation initiation codons or stop codons. Thirty-two sites were commonly observed in the hornwort Anthoceros angustus. This finding suggests that the enigmatic bryophyte Takakia is closely related to hornworts with respect to RNA editing events.

Base Sequence↗
PubMed1992

A TBP complex essential for transcription from TATA-less but not TATA-containing RNA polymerase III promoters is part of the TFIIIB fraction.

The TATA box-binding protein TBP directs transcription by all three eukaryotic RNA polymerases. In mammalian cells, TBP is found in at least three different complexes: SL1, D-TFIID, and B-TFIID. While SL1 and D-TFIID are involved in RNA polymerase I and II transcription, respectively, no unique function has been assigned to the B-TFIID complex. Here we show that the TFIIIB fraction required for RNA polymerase III transcription contains two separable components, one of which is a TBP-containing complex that may correspond to B-TFIID. For transcription of TATA-less RNA polymerase III genes such as the VAI, 5S, and 7SL genes, this complex cannot be replaced by either TBP alone or the D-TFIID complex. In contrast, TBP alone is active for basal transcription from the TATA-containing U6 promoter. This indicates different requirements for recruiting TBP to TATA-less and TATA-containing RNA polymerase III promoters.

Base Sequence↗
PubMed1993

Analysis of deletions and thermosensitive mutations in Rous sarcoma virus gag protein p10.

Rous sarcoma virus protein p10 is a gag component of the virion present in stoichiometric amount but of unknown function. To characterize this protein, a series of mutants of p10 with linker insertions or deletions was generated by site-directed mutagenesis of a cloned proviral DNA. The deletions and two of the linkers insertions, which disrupted proline pairs, reduced the yield of virus particles upon transfection. These two linker insertion mutants were moreover thermosensitive for this phenotype, producing fewer virus particles at 41 degrees C than at 36 degrees C. Examination of the intracellular viral proteins demonstrated that for all mutants, the amount of gag precursor was similar to the wild-type level. Moreover, the amount of mature gag CA that could be detected by this analysis was similar between each of the mutants and the wild type. This finding suggests that the transport of gag to the membrane and the initial stages of maturation were not affected by the mutations. The virus particles contained normal amounts of active reverse transcriptase, showing that the gag-pol polyprotein was incorporated and cleaved properly. Viral RNA was quantitatively and qualitatively similar in mutant and wild-type virions. However, the infectivity of the mutants virions differed; one of the thermosensitive linker insertions that had no effect on particle production at 36 degrees C was nevertheless noninfectious at that temperature. Together, these data suggest that the p10 protein is involved in a late steps of virus maturation, possibly budding, and perhaps also in an early event of viral infection.

Amino Acid Sequence↗
PubMedDate not supplied

The interaction between Bacillus subtilis sigma-A (sigma A) factor and RNA polymerase with promoters.

The P2 promoter from Bacillus subtilis sigma-A (sigma A) operon and the strong phi 29 phage G3b promoter were used to study their interactions with free sigma A and with RNA polymerase holoenzymes (E sigma A and E sigma 70). No binding of free sigma A to the tested promoters was observed, suggesting that the B subtilis free sigma A does not bind promoter by itself for the initiation of RNA transcription. Different footprints of B subtilis RNA polymerase holoenzyme (E sigma A) on the P2 and G3b promoters were detected. The footprint on the P2 promoter is mainly in the -10 downstream region of the bottom strand (noncoding strand) DNA and limited on the top strand (coding strand), whereas the footprints on both strands of the G3b promoter are very clear. These results suggest that the footprint regions of RNA polymerase on a promoter and the strength of its binding to the promoter depend on the properties of the specific promoter DNA sequence. It also suggests that the -10 and its downstream regions are more important than the -35 region for the formation of the E sigma A-P2 promoter open complex. Footprints of B subtilis E sigma A and E coli E sigma 70 on the same G3b promoter are very similar on the top strand but different on the bottom strand, with the footprint being about 17 bases wider (-4 to +13) in the case of E coli E sigma 70. Since this region contains most of the bases involved in promoter DNA melting, we suggest that E coli and B subtilis RNA polymerases have different efficiency in forming the open complex with heterologous promoter DNA during initiation of transcription.

Bacillus Phages↗
PubMed1999

Reevaluation of the promoter structure of the class 3 flagellar operons of Escherichia coli and Salmonella.

Flagellar class 3 operons of Escherichia coli and Salmonella are transcribed by RNA polymerase containing sigma 28. The consensus sequence of the sigma 28-dependent promoters was believed to be TAAA N15 GCCGATAA. In this study, we found that the E. coli genome contains a large number of sequences homologous to this consensus. However, we showed that they do not always exert a sigma 28-dependent promoter activity. We compare more carefully the sequences of the class 3 flagellar promoters and propose a revised structure of the sigma 28-dependent promoters as TAAAGTTT N11 GCCGATAA.

Base Sequence↗
PubMed1994

Expression in Escherichia coli: production and purification of both subunits of the human general transcription factor TFIIE.

Both subunits of the human class II transcription factor TFIIE (rTFIIE alpha and rTFIIE beta) have been overexpressed in Escherichia coli at 26 degrees C using a T7 RNA polymerase expression system and further purified to apparent homogeneity. As in this system rTFIIE alpha was poorly expressed and copurified with a truncated form, we expressed rTFIIE alpha as a fusion protein. These overexpressed subunits of TFIIE are similar to the endogenous subunits according to the following criteria: molecular weight, microsequencing, and transcription activity.

Affinity Labels↗
PubMed2004

A type of nucleotide motif that distinguishes tobamovirus species more efficiently than nucleotide signatures.

The complete genomic sequences of forty-eight tobamoviruses were classified and found to form at least twelve species clusters. Individual species were not conveniently defined by 'nucleotide signatures' (i.e. strings of one or more nucleotides unique to a taxon) as these were scattered sparsely throughout the genomes and were mostly single nucleotides. By contrast all the species were concisely and uniquely distinguished by short nucleotide motifs consisting of conserved genus-specific sites intercalated with variable sites that provided species-specific combinations of nucleotides (nucleotide combination motifs; NC-motifs). We describe the procedure for finding NC-motifs in a convenient and phylogenetically conserved region of the tobamovirus RNA polymerase gene, the '4404-50 motif'. NC-motifs have been found in other sets of homologous sequences, and are convenient for use in published taxonomic descriptions.

Conserved Sequence↗
PubMed1994

Study on replicase (subunit) gene of papaya ringspot virus cloning, sequencing and construction of higher plant expression vector.

The ds-cDNA was synthesized using genomic RNA of papaya ringspot virus (PRSV) as template. The blunt-ended cDNA was cloned into the EcoRV site of vector pBluescript SK. From the recombinants, the NIb gene of PRSV was obtained, and its complete sequence was also determined. After the NIb gene was modified through PCR, a binary vector of PRSV-NIb gene was constructed for its expression in higher plants.

Amino Acid Sequence↗
PubMed1997

Rapid mutagenesis and purification of phage RNA polymerases.

We have developed plasmid-based expression systems that encode modified forms of T7 RNA polymerase (RNAP) having 6-12 histidine residues fused to the amino terminus. The histidine-tagged RNAPs (His-T7 RNAPS) are indistinguishable from the wild-type (WT) enzyme in nearly all biochemical assays. Similar plasmids that encode His-tagged T3 and SP6 RNAPs have also been constructed. To facilitate site-directed mutagenesis of the RNAP gene, the size of the target plasmid was minimized by using T7 RNAP itself as a selectable marker. BL21 (DCAT4) cells (which carry a chromosomal copy of the chloramphenicol acetyltransferase cat gene under control of a T7 promoter) are resistant to chloramphenicol when functional T7 RNAP is expressed, thus allowing the selection and maintenance of the target plasmid in these cells. Mutagenesis is accomplished by denaturing the plasmid, annealing mutagenic DNA primers, and repairing the plasmid with T4 DNA polymerase. Two DNA primers are used: one corrects a defect in the bla gene, the other introduces the desired mutation into the RNAP gene; 30-85% of the ampicillin-resistant transformants carry the desired mutation in the RNAP gene. By using BL21 (DCAT4) cells as a recipient for transformation the functional integrity of the RNAP gene may conveniently be monitored by assessing the level of chloramphenicol resistance in vivo. Methods for rapid, simultaneous purification of multiple samples of modified (His-tagged) and conventional RNAPs are described. Together, these developments greatly enhance our ability to characterize this important class of enzymes.

Amino Acid Sequence↗
PubMed1992

The phage lambda gene Q transcription antiterminator binds DNA in the late gene promoter as it modifies RNA polymerase.

The bacteriophage lambda gene Q transcription antiterminator modifies RNA polymerase during an extended pause in elongation at nt +16 and +17 of the phage late gene promoter transcript. We show here that Q binds a specific DNA site between the -10 and -35 elements of the promoter as it interacts with the enzyme. We show that the pause must reflect a specialized elongation structure that is receptive to modification by Q, because Q does not bind to RNA polymerase stopped artificially after transcribing 16 nt of mutant DNA that does not encode the natural pause. Footprinting shows that RNA polymerase in the paused complex makes distinctive interactions with DNA in the region where Q binds; binding of Q, in turn, changes the footprint both at the Q-binding site and in the transcription bubble. Binding of Q to the paused transcription complex is stabilized by the transcription factor NusA, as expected from the dependence of lambda Q-mediated antitermination on NusA.

Bacteriophage lambda↗
PubMed1988

Isolation of a Bacillus thuringiensis RNA polymerase capable of transcribing crystal protein genes.

We report the isolation of an RNA polymerase from sporulating cells of Bacillus thuringiensis subsp. kurstaki HD-1-Dipel that directs transcription from the promoter region of an insecticidal crystal protein gene. The core components of this RNA polymerase are associated with a polypeptide that has an apparent mass of 35 kDa. Neither RNA polymerase holoenzyme isolated from vegetative B. thuringiensis, nor the core derived from this enzyme, is capable of transcribing from the crystal protein gene promoter region; the addition of gel-purified 35-kDa polypeptide to the core reconstitutes the specific transcribing capability. The reconstituted enzyme does not direct transcription from the promoters for the ctc or spoVG genes of Bacillus subtilis; however, this form of RNA polymerase does direct transcription from a promoter for the 27-kDa crystal protein of B. thuringiensis subsp. israelensis and from a promoter for a 29-kDa polypeptide present in cuboidal crystals of B. thuringiensis subsp. kurstaki HD-1. We propose a tentative consensus sequence based on the alignment of the three B. thuringiensis promoters. This consensus sequence is different from consensus sequences reported for promoters recognized by enzymes containing other sigma subunits, suggesting that the 35-kDa polypeptide is an unusual sigma subunit.

Bacillus thuringiensis↗