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PubMed2005

DIRS-1 and the other tyrosine recombinase retrotransposons.

DIRS-1 is a retroelement from the slime mold Dictyostelium discoideum. Until recently only two related retrotransposons had been described: PAT from the nematode Panagrellus redivivus and Prt1 from the zygomycete fungus Phycomyces blakesleeanus. Analyses of the reverse transcriptase sequences encoded by these three elements suggested that they were closely related to each other and more distantly related to the Ty3/gypsy Long Terminal Repeat (LTR) retroelements. They have several unusual structural features that distinguish them from typical LTR elements. For instance, they each encode a tyrosine recombinase (YR), but not a DDE-type integrase or an aspartic protease. Although the DIRS-1-related elements are bordered by terminal repeats these differ from typical LTRs in a number of ways. In DIRS-1, for example, the terminal repeats are inverted (complementary), non-identical in sequence, and the outer edges of the terminal sequences are repeated (adjacent to each other) in the internal region. PAT has so-called "split" direct repeats in which the unrelated terminal sequences appear as direct repeats adjacent to each other in the internal region. The only repetition displayed by Prt1 is the presence of short inverted terminal repeats, but the sequenced copy of this element is believed to be a truncated version of an element with a structure resembling DIRS-1. The unusual structure of the terminal repeats of the DIRS1-like elements appears to be related to their replication via free circular intermediates. Site-specific recombination is believed to integrate the circle without creating duplications of the target sites. In recognition of these important distinctions it is proposed that the retrotransposons that encode tyrosine recombinases be called the tyrosine recombinase (or YR) retrotransposons. Recently a large number of additional YR retrotransposons have been described, including elements from fungi (zygomycetes and basidiomycetes), plants (green algae) and a wide range of animals including nematodes, insects, sea urchins, fish and amphibia, while remnants of elements related to DIRS-1 occur in the human genome. The complete set of YR retrotransposons can be divided into two major groups, the DIRS elements and the Ngaro elements, the two groups forming distinct clades on phylogenetic trees based on alignments of RT/RH and recombinase sequences, and also having some structural distinctions. A third group of transposable elements, which we call Cryptons, also carry tyrosine recombinases. These elements do not encode a reverse transcriptase and so are believed to be DNA transposons not retrotransposons. They have been detected in several pathogenic fungi, including the basidiomycete Cryptococcus neoformans, and the ascomycetes Coccidioides posadasii and Histoplasma capsulatum. Sequence comparisons suggest that the Crypton YRs are related to those of the YR retrotransposons. We suggest that the YR retrotransposons arose from the combination of a Crypton-like YR DNA transposon and the RT/RH encoding sequence of a retrotransposon.

Amino Acid Sequence↗
PubMedDate not supplied

[Reaction of pyrophosphorolysis catalyzed by Escherichia coli RNA polymerase].

E. coli RNA polymerase is shown to be capable of catalyzing consecutive DNA-dependent pyrophosphorolysis of RNA in the presence of inorganic pyrophosphate and Mg2+. Active ternary complex of the enzyme with DNA and nascent RNA is needed for the reaction, the mixure of all the components can not carry out pyrophosphorolysis. The reaction was realized in the absence of added nucleoside triphosphates. Nucleoside triphosphates are low molecular mass products of the reaction. The rate of pyrophosphorolysis of the RNA synthesised for the AI promoter of the DNA of wild type T7 phage and delta D III T7 mutant phage was followed as a function of primary structure of the DNA, temperature, ionic strength and inorganic pyrophosphate concentration. The relative rate pyrophosphorolysis for particular nucleotides in different regions of the RNA can differ by several orders of magnitude depending on the primary structure of the RNA that undergoes pyrophosphorolysis. Ternary complex containing partially pyrophosphorilised RNA is active on the RNA synthesis when pyrophosphate is removed and nucleoside triphosphates are added to the reaction mixture. RNA as short as 70-8 nucleotides long can be produced at the conditions used. It seems that efficient dissociation in this region of RNA limits the pyrophosphorolysis to proceed up to the 5' end of RNA. Ternary complex of RNA polymerase with nascent RNA and DNA is shown to undergo site specific dissociation. The specificity of the dissociation is shown to be a function of the primary structure of RNA and the direction of the reaction. Dissociation occurs at different places along RNA sequence when the RNA is synthesised and when it is pyrophosphorilised.

Base Sequence↗
PubMed2004

Surfaces of Spo0A and RNA polymerase sigma factor A that interact at the spoIIG promoter in Bacillus subtilis.

In Bacillus subtilis, the DNA binding protein Spo0A activates transcription from two classes of promoters, those used by RNA polymerase containing the primary sigma factor, sigma(A) (e.g., spoIIG), and those used by RNA polymerase containing the secondary sigma factor, sigma(H) (e.g., spoIIA). Several single amino acid substitutions in region 4 of sigma(A) define positions in sigma(A) that are specifically required for Spo0A-dependent promoter activation. Similarly, several single amino acid substitutions in Spo0A define positions in Spo0A that are required for sigma(A)-dependent promoter activation but not for other functions of Spo0A. It is unknown whether these amino acids in Spo0A interact directly with those in region 4 of sigma(A) or whether they interact with another subunit of RNA polymerase to effect promoter activation. Here we report the identification of a new amino acid in region 4 of sigma(A), arginine at position 355 (R355), that is involved in Spo0A-dependent promoter activation. To further investigate the role of R355, we used the coordinates of Spo0A and sigma region 4, each in complex with DNA, to build a model for the interaction of sigma(A) and Spo0A at the spoIIG promoter. We tested the model by examining the effects of amino acid substitutions in the putative interacting surfaces of these molecules. As predicted by the model, we found genetic evidence for interaction of R355 of sigma(A) with glutamine at position 221 of Spo0A. These results appear to define the surfaces of Spo0A and sigma(A) that directly interact during activation of the spoIIG promoter.

Amino Acid Substitution↗
PubMed1977

Nucleotide sequence at the 5' terminus of the avian sarcoma virus genome.

Transcription of DNA from the RNA genome of avian sarcoma virus by RNA-directed DNA polymerase in vitro initiates on a primer (tRNATrp) located near the 5'-terminus of the viral genome. One of the major products of transcription is a single-stranded DNA chain complementary to a sequence of 101 nucleotides immediately distal to the site of initiation of DNA synthesis. We have determined the complete nucleotide sequence of this transcribed chain for the Prague strain of avian sarcoma virus, a partial sequence of the transcribed chain for the Bratislava 77 strain of avian sarcoma virus, and the sequence of a DNA transcript that is shorter than the transcribed single-stranded chain. Our data define the location of tRNATrp on the genome of avian sarcoma virus and provide the sequence of 119 nucleotides at the 5'-terminus of the genome. Portions of this sequence may be involved in the binding of RNA-directed DNA polymerase, the initiation of translation from viral messenger RNA, the extension of RNA-directed DNA synthesis from the 5'- to the 3'-terminus of viral RNA, and the integration of viral DNA into the host genome.

Avian Sarcoma Viruses↗
PubMed2002

Role of an essential triloop hairpin and flanking structures in the 3' untranslated region of Alfalfa mosaic virus RNA in in vitro transcription.

The minus-strand promoter of Alfalfa mosaic virus (AMV), a tripartite plant virus belonging to the family Bromoviridae, is located within the 3'-terminal 145 nucleotides (nt), which can adopt a tRNA-like structure (TLS). This contrasts with the subgenomic promoter for RNA4 synthesis, which requires approximately 40 nt and forms a single triloop hairpin. Detailed analysis of the minus-strand promoter now shows that a similar triloop hairpin, hairpin E (hpE), is crucial for minus-strand synthesis. The loop sequence of hpE appeared to not be essential for RNA synthesis, whereas the identity and base-pairing capability of bases below the triloop were indeed essential. Reducing the size of the bulge loop of hpE triggered transcription from an internal site similar to the process of subgenomic transcription. Similar effects were observed when deleting (part of) the TLS, suggesting that tertiary contacts between hpE and the TLS prevent internal initiation. The data indicate that the minus-strand promoter hpE and the subgenomic promoter hairpin are equivalent in binding the viral polymerase. We propose that the major role of the TLS is to enforce the initiation of transcription by polymerase at the very 3' end of the genome.

3' Untranslated Regions↗
PubMed1996

Transcription activation by the bacteriophage Mu Mor protein requires the C-terminal regions of both alpha and sigma70 subunits of Escherichia coli RNA polymerase.

Middle transcription of bacteriophage Mu requires Escherichia coli RNA polymerase and a Mu-encoded protein, Mor. Consistent with these requirements, the middle promoter, Pm, has a -10 hexamer but lacks a recognizable -35 hexamer. Interactions between Mor and RNA polymerase were studied using in vitro transcription, DNase I footprinting, and the yeast interaction trap system. We observed reduced promoter activity in vitro using reconstituted RNA polymerases with C-terminal deletions in alpha or sigma70. As predicted if alpha were binding to Pm, we detected a polymerase-dependent footprint in the -60 region. Reconstituted RNA polymerases containing Ala substitutions in the alpha C-terminal domain were used to assay Mor-dependent transcription from Pm in vitro. The D258A substitution and alpha deletion gave large reductions in activation, whereas the L262A, R265A, and N268A substitutions caused smaller reductions. The interaction trap assay revealed weak interactions between Mor and both alpha and sigma70; consistent with a key role of alpha-D258, the D258A substitution abolished interaction, whereas the R265A substitution did not. We propose that: (i) alpha-D258 is a Mor "contact site"; and (ii) residues Leu-262, Arg-265, and Asn-268 indirectly affect Mor-polymerase interaction by stabilizing the ternary complex via alpha-DNA contact.

Base Sequence↗
PubMed1995

Interference with bacteriophage phi 6 genomic RNA packaging by hairpin structures.

Bacteriophage phi 6 has a genome of three segments of double-stranded RNA enclosed in a procapsid composed of four different proteins. The preformed procapsid is capable of packaging plus-strand transcripts of the genomic segments in an in vitro reaction. Minus-strand synthesis within the procapsid then results in the production of the double-stranded RNA genome. When plus-strand transcripts contain strong hairpin structures near the 3' ends, they are subject to heterologous recombination to remove the hairpins. We now find that the sequences bounded by the hairpins as well as those 3' to them are excluded from particles in packaging reactions. This finding implies that packaging occurs from the 5' end and that the explanation for the facilitation of recombination by the hairpin structures is the lack of entry of the 3' ends rather than a difficulty of progressing through the hairpin by the phage polymerase. Packaging of segment M is dependent on the packaging of segment S. An S segment containing a strong hairpin is able to facilitate the packaging of segment M. This result implies that there is more than one entry pore into the procapsid.

Bacteriophage phi 6↗
PubMed1994

Genetic and physiological studies of Bacillus subtilis sigma A mutants defective in promoter melting.

The Bacillus subtilis sigA gene encodes the primary sigma factor of RNA polymerase and is essential for cell growth. We have mutated conserved region 2.3 of the sigma A protein to substitute each of seven aromatic amino acids with alanine. Several of these aromatic amino acids are proposed to form a melting motif which facilitates the strand separation step of initiation. Holoenzymes containing mutant sigma factors recognize promoters, but some are defective for DNA melting in vitro. We have studied the ability of each mutant sigma factor to support cell growth by gene replacement and complementation. The two region 2.3 mutants least impaired in promoter melting in vitro (Y180A and Y184A) support cell growth in single copy, although the Y184A allele imparts a slow-growth phenotype at low temperatures. A strain expressing only the Y189A variant of the sigma A protein, known to be defective in DNA melting in vitro, grows very slowly and is altered in its pattern of protein synthesis. Only the wild-type and Y180A sigma A proteins efficiently complement a temperature-sensitive allele of sigA. Overexpression of three of the sigma A proteins defective for promoter melting in vitro (Y189A, W192A, and W193A) leads to a decrease in RNA synthesis and cell death. These results indicate that mutations which specifically impair DNA melting in vitro also impair sigma function in vivo and therefore support the hypothesis that sigma plays an essential role in both DNA melting and promoter recognition.

Alleles↗
PubMed1978

Semi-conservative transcription in particles of a double-stranded RNA mycovirus.

During transcription in vitro catalysed by the virion RNA polymerase of Aspergillus foetidus virus AfV-S in the presence of tritiated UTP, the virus double-stranded RNA becomes labelled in one strand, which has the same sequence as the single-stranded RNA transcripts produced. Most of the label incorporated into double-stranded RNA could be chased into single stranded RNA by further reaction with excess unlabelled nucleoside triphosphates. In reactions containing tritiated UTP the single-stranded RNA transcripts released after the first round of transcription were unlabelled. It is concluded that transcription in virions of AfV-S occurs by displacement of one of the strands of double-stranded RNA by the RNA strand being newly synthesised i.e. the reaction is semi-conservative with respect to double-stranded RNA.

Aspergillus↗
PubMed1990

Regulation of transcriptional initiation in yeast mitochondria.

We have investigated in vitro transcriptional initiation by purified yeast mitochondrial RNA polymerase using a variety of previously described promoter variants and dinucleotides corresponding to the first two transcript nucleotides. Regardless of the actual nucleotides that occupy the first two transcript positions, the rate of initiation increases with increasing concentrations of the first two ribonucleoside triphosphates up to 125 microM whereas elongation is carried out optimally with less than 10 microM. Under normal in vitro transcription conditions, mitochondrial RNA polymerase only employs the in vitro start site (+1 position), again without regard to the nucleotide at the position. Even with initiator dinucleotide monophosphates as primers, the polymerase is only capable of initiating transcription at this position and one other, i.e. 1 base upstream (-1). Dinucleotides enhance transcription from partially active variant promoters (mutations around the initiation sites -3, -1, +1, +2), suggesting that these mutations reduce transcription by their effects on initiation. In contrast, inactive promoters (-7C, -6G, -4A, and -2A) are not active in the presence of initiating dinucleotide. We suggest that dinucleotides may function in one of three ways: (i) bypassing the energy barrier in forming the first internucleotide bond; (ii) stabilizing the initiation complex; or (iii) accelerating promoter clearance.

Base Sequence↗
PubMed2006

Mutational analysis of an extracytoplasmic-function sigma factor to investigate its interactions with RNA polymerase and DNA.

The extracytoplasmic-function (ECF) family of sigma factors comprises a large group of proteins required for synthesis of a wide variety of extracytoplasmic products by bacteria. Residues important for core RNA polymerase (RNAP) binding, DNA melting, and promoter recognition have been identified in conserved regions 2 and 4.2 of primary sigma factors. Seventeen residues in region 2 and eight residues in region 4.2 of an ECF sigma factor, PvdS from Pseudomonas aeruginosa, were selected for alanine-scanning mutagenesis on the basis of sequence alignments with other sigma factors. Fourteen of the mutations in region 2 had a significant effect on protein function in an in vivo assay. Four proteins with alterations in regions 2.1 and 2.2 were purified as His-tagged fusions, and all showed a reduced affinity for core RNAP in vitro, consistent with a role in core binding. Region 2.3 and 2.4 mutant proteins retained the ability to bind core RNAP, but four mutants had reduced or no ability to cause core RNA polymerase to bind promoter DNA in a band-shift assay, identifying residues important for DNA binding. All mutations in region 4.2 reduced the activity of PvdS in vivo. Two of the region 4.2 mutant proteins were purified, and each showed a reduced ability to cause core RNA polymerase to bind to promoter DNA. The results show that some residues in PvdS have functions equivalent to those of corresponding residues in primary sigma factors; however, they also show that several residues not shared with primary sigma factors contribute to protein function.

Amino Acid Sequence↗
PubMed1998

Rapid isolation of RNA polymerase from sporulating cells of Bacillus subtilis.

A highly ordered program of temporal and spatial gene activation during sporulation in Bacillus subtilis is governed by the principal RNA polymerase, and RNA polymerases containing at least five developmental sigma factors appearing successively during sporulation. This report describes a rapid procedure for extracting RNA polymerase from sporulating B. subtilis cells, which involves the construction of hexahistidine tagged beta' subunit of RNA polymerase and the isolation of RNA polymerase holoenzyme with Ni2+-NTA resin. In in vitro transcription of various promoters with the RNA polymerase thus purified, we observed the temporal change of each RNA polymerase activity during sporulation. This procedure enables isolation of RNA polymerase within 4h, starting with cell pellets. Our results indicated that a principal sigma factor, sigmaA, could be detected in a holoenzyme form during all the stages of growth and sporulation, while the other sigma factors sigmaH, sigmaE, sigmaF, sigmaG, and sigmaK involved in sporulation could be detected sequentially during sporulation. Moreover, Spo0A, the central transcription factor of commitment to sporulation, was also co-purified with RNA polymerase at early stages of sporulation.

Bacillus subtilis↗
PubMed1996

RNA-DNA hybrid formation at the human mitochondrial heavy-strand origin ceases at replication start sites: an implication for RNA-DNA hybrids serving as primers.

Critical elements of a mammalian mitochondrial DNA heavy-strand replication origin include a promoter and three downstream conserved sequence blocks (CSBIII, CSBII and CSBI). We found recently that a stable and persistent RNA-DNA hybrid forms during in vitro transcription at Saccharomyces cerevisiae mitochondrial origins; hybrid formation was dependent on the conserved CSBII element. We report here that during in vitro transcription with human mitochondrial RNA polymerase, stable and persistent RNA-DNA hybrid formation is also evident at the human mitochondrial heavy-strand origin. As predicted, hybrid formation was dependent on the GC-rich CSBII element. The human RNA-DNA hybrids terminate within or downstream of CSBI at locations implicated in initiation of mitochondrial DNA replication. Interestingly, efficient hybrid formation in the human system is influenced by sequence 5' to the RNA-DNA hybrid, including the CSBIII element. These results suggest that the RNA-DNA hybrids formed during transcription across the mitochondrial DNA heavy-strand origin provide RNA primers for initiation of mitochondrial DNA replication.

Base Sequence↗
PubMed1992

A new method for reconstituting influenza polymerase and RNA in vitro: a study of the promoter elements for cRNA and vRNA synthesis in vitro and viral rescue in vivo.

The influenza RNA polymerase is known to catalyse three distinct copying activities: (i) transcription of minus-sense virion RNA (vRNA) into mRNA, (ii) transcription of vRNA into full-length complementary RNA (cRNA), and (iii) transcription of cRNA to vRNA. Ever since the discovery of the conserved 13 and 12 long sequences at each end of all the influenza RNA segments, these have been good candidates for promoters of transcription. By devising a new, simple method for preparing influenza polymerase complex capable of transcribing in vitro added short model RNA templates without interference from endogenous viral RNA, we have now tested the promoter hypothesis. We conclude that the 13 long and the 12 long 3' conserved sequences of cRNA and vRNA of influenza A virus are by themselves sufficient to promote vRNA and cRNA synthesis in vitro. Using our new method, we also show that chloramphenicol acetyl transferase (CAT) activity can be detected in MDBK (bovine kidney) cells, after transfection of influenza polymerase assembled with a negatively stranded CAT RNA, even in the absence of helper virus. As in a previously described method (Luytjes et al., 1989), CAT activity is amplified by helper virus and can be rescued in infectious recombinant virus.

Base Sequence↗
PubMed1995

Ribozymes expressed within the loop of a natural antisense RNA form functional transcription terminators.

As an initial step towards developing a widely applicable system for expressing small ribozymes (Rz) in various cell types using T7 RNA polymerase, we have replaced the loop domain of a natural prokaryotic antisense RNA (RNAout from Tn10), with hammerhead (Hh) Rz. RNAout was chosen, because the stem of its secondary structure gives it an unusually long half life in Escherichia coli which should also confer in vivo stability to small RNA sequences expressed within its loop domain. In order to define the 3' end of the Rz-RNAout chimeric RNAs, a poly(U) tract was inserted just 3' of the RNAout stem. Molecular analysis indicates that these RNAs function both as transcription terminators and Rz. In addition, the RNAs are stable in E. coli and can be expressed in mammalian cells. These results show that certain characteristics of a naturally evolved RNA, RNAout in this case, can be used to provide additional functions to short RNAs containing Hh Rz without disrupting the enzymatic activity of the Rz.

Animals↗
PubMed2003

Purification and in vitro characterization of the Serratia marcescens NucC protein, a zinc-binding transcription factor homologous to P2 Ogr.

NucC is structurally and functionally homologous to a family of prokaryotic zinc finger transcription factors required for late gene expression in P2- and P4-related bacteriophages. Characterization of these proteins in vitro has been hampered by their relative insolubility and tendency to aggregate. We report here the successful purification of soluble, active, wild-type NucC protein. Purified NucC exhibits site-specific binding to a conserved DNA sequence that is located upstream of NucC-dependent Serratia marcescens promoters and the late promoters of P2-related phages. This sequence is sufficient for binding of NucC in vitro. NucC binding to the S. marcescens nuclease promoter P(nucA) and to the sequence upstream of the P2 late promoter P(F) is accompanied by DNA bending. NucC protects about 25 nucleotides of the P(F) upstream region from DNase I digestion, and RNA polymerase protects the promoter region only in the presence of NucC. Template DNA, RNA polymerase holoenzyme, and purified NucC are the only macromolecular components required for transcription from P(F) in vitro.

Bacterial Proteins↗