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At least 757 records · Page 42Linked to original sources
PubMed1994

Identification of a DNA-contacting surface in the transcription factor sigma-54.

The transcription factor sigma-54 (sigma 54) is a sequence-specific DNA-binding protein that directs RNA polymerase to a particular class of promoter. The interaction of sigma 54 with promoter DNA has been analysed by protein-DNA crosslinking and enzymatic and chemical proteolysis. Direct physical evidence for a DNA-contacting surface within the carboxy-terminal one-third of the protein has been obtained. This region of sigma 54 is likely to be close to the surface of the protein, and contacts DNA when either sigma 54 or the sigma 54-holoenzyme bind specifically to promoter DNA. The amino-terminal region of sigma 54 appears to be highly susceptible to proteolysis, and its integrity influences the accessibility towards proteolysis of a second region of sigma 54, which includes the DNA-contacting surface. Thus the amino-terminal region of sigma 54 may have a role in influencing its DNA-binding properties, the major determinants of which appear to reside in the carboxy-terminal one-third of the protein.

Bacterial Proteins↗
PubMed1995

Sequences in the preC region of duck hepatitis B virus affect pregenomic RNA accumulation.

The pregenomic RNA of hepadnaviruses serves as both the mRNA for the core and polymerase proteins and the RNA template for reverse transcription. We have identified a region in the duck hepatitis B virus pregenomic RNA transcription unit that is critical for the accumulation of this transcript. This 85-nt region, termed alpha, is located within the preC region; deletion of alpha results in drastically reduced steady-state levels of pregenomic RNA. This effect is not due to reduction in transcription initiation or to enhancement of premature polyadenylation at the 5' copy of the viral poly(A) signal. However, this phenotype is suppressed by deletion of a second, larger region (beta) located ca. 1 kb downstream. The activity of the alpha element is tissue- and species-nonspecific; however, it displays absolute orientation-dependence and its activity is influenced by its position within the transcript. Models for its action are discussed.

Animals↗
PubMed1992

A patient with von Willebrand's disease characterized by a compound heterozygosity for a substitution of Arg854 by Gln in the putative factor-VIII-binding domain of von Willebrand factor (vWF) on one allele and very low levels of mRNA from the second vWF allele.

We describe a patient with a lifelong bleeding disorder previously classified as von Willebrand's disease (vWD) type I. The factor VIII (FVIII) level in this patient was disproportionately low and we showed that this was due to a decreased factor VIII binding capacity of her vWF. To characterize the molecular defect in this type of vWD, a cDNA-dependent polymerase chain reaction (PCR) amplification was performed using platelet RNA as a template. Direct sequencing of the amplified fragment, which encodes for the FVIII-binding domain, showed a single nucleotide change in exon 20 at codon 854, resulting in the substitution of CAG glutamine (Gln) for CGG arginine (Arg). At the level of the cDNA only the mutated sequence was found, whereas at genomic DNA level the patient was heterozygous for this mutation. This patient is therefore a compound heterozygote for a point mutation resulting in a FVIII-binding defect and a vWF allele with low transcript levels.

Adult↗
PubMed1999

Cra-dependent transcriptional activation of the icd gene of Escherichia coli.

The icd gene of Escherichia coli, encoding isocitrate dehydrogenase, was shown to be expressed from two different promoters: the previously identified icd P1 and a newly detected second promoter, icd P2, whose expression is positively regulated by the catabolite repressor-activator protein Cra, formerly called FruR. In each case, we determined the mRNA start site by primer extension analysis of in vivo transcripts and examined the interaction of the icd control region with either RNA polymerase or Cra. We observed that (i) the Cra factor binds to and activates transcription from a site centered at position -76.5 within the icd P2 promoter region and (ii) three particular mutations in the C-terminal end of the alpha subunit of RNA polymerase (L262A, R265A, and N268A) considerably diminish transcription initiating from the icd P2 promoter, as shown by in vitro experiments performed in the presence of mutant RNA polymerases carrying Ala substitutions.

Amino Acid Substitution↗
PubMed1994

Functional reconstitution of wild-type and mutant Tetrahymena telomerase.

Telomerase is a ribonucleoprotein that catalyzes telomere elongation in vitro and in vivo. The 159-nucleotide RNA component of Tetrahymena telomerase contains the sequence 5'-CAACCCCAA-3' ("template region"), which serves as a template for the addition of the sequence d(TTGGGG)n to Tetrahymena telomeres. To dissect the Tetrahymena telomerase enzyme mechanism, we developed a functional in vitro reconstitution assay. After removal of the essential telomerase RNA by micrococcal nuclease digestion of partially purified telomerase, the addition of in vitro-transcribed telomerase RNA reconstituted telomerase activity. The reconstituted activity was processive and showed the same primer specificities as native telomerase. Mutants in the RNA template region were tested in reconstitution assays to determine the role of the residues in this region in primer recognition and elongation. Two template mutants, encoding the sequences 5'-UAACCCCAA-3' and 5'-UAACCCUAA-3', specified the incorporation of dATP into the sequence d(TTAGGG). Telomerase reconstituted with a template mutant encoding the sequence 5'-CAACCCUAA-3' did not specify dATP incorporation and elongation by this mutant was not terminated by the addition of ddATP. In addition, a template mutant encoding the sequence 5'-CGGCCCCAA-3' specified the incorporation of ddCTP but not ddTTP while a mutant encoding the sequence 5'-CAACCCCGG-3' specified the incorporation of ddTTP but not ddCTP. These data suggest that only the most 5' six residues of the template region dictate the addition of telomeric repeats.

Animals↗
PubMed1995

Isolation and characterization of a retroelement from B chromosome (PSR) in the parasitic wasp Nasonia vitripennis.

Molecular characterization of the paternal-sex-ratio (PSR) chromosome in Nasonia vitripennis (Hymenoptera: Pteromalidae) has led to the isolation of a dispersed repetitive element. The element is a LTR-containing retrotransposon which has been named NATE (NAsonia Transposable Element). NATE has direct terminal repeats and has an internal amino acid sequence similar to reverse transcriptases of other retroelements. Phylogenetic analysis indicates NATE is a member of the Gypsy/Ty3 group of retrotransposons, and represents the first isolated from Hymenoptera. Five closely related copies of NATE were isolated from the PSR chromosome, but cross-hybridizing elements were not detected on the autosomes of N. vitripennis. Strongly cross-hybridizing elements were, however, detected in two other Nasonia species. This observed distribution of NATE is interesting, because the supernumerary PSR chromosome may be derived from the genome of a sibling species of N. vitripennis.

Amino Acid Sequence↗
PubMed2000

Constitutive expression of a transcription termination factor by a repressed prophage: promoters for transcribing the phage HK022 nun gene.

Lysogens of phage HK022 are resistant to infection by phage lambda. Lambda resistance is caused by the action of the HK022 Nun protein, which prematurely terminates early lambda transcripts. We report here that transcription of the nun gene initiates at a constitutive prophage promoter, P(Nun), located just upstream of the protein coding sequence. The 5' end of the transcript was determined by primer extension analysis of RNA isolated from HK022 lysogens or RNA made in vitro by transcribing a template containing the promoter with purified Escherichia coli RNA polymerase. Inactivation of P(Nun) by mutation greatly reduced Nun activity and Nun antigen in an HK022 lysogen. However, a low level of residual activity was detected, suggesting that a secondary promoter also contributes to nun expression. We found one possible secondary promoter, P(Nun)', just upstream of P(Nun). Neither promoter is likely to increase the expression of other phage genes in a lysogen because their transcripts should be terminated downstream of nun. We estimate that HK022 lysogens in stationary phase contain several hundred molecules of Nun per cell and that cells in exponential phase probably contain fewer.

Bacteriophage lambda↗
PubMed1997

A mutant T7 RNA polymerase that is defective in RNA binding and blocked in the early stages of transcription.

We have identified a mutation (E148A) in T7 RNA polymerase (RNAP) that results in an enzyme which aborts transcription primarily when the nascent RNA achieves a length of 5 nt. This phenomenon is observed at a consensus promoter, but is even more strongly observed at promoters that are altered in the initiation region. Although the abortive product is of a fixed length (5 nt), the positions of the base substitutions in the initiation region that enhance this effect do not appear to be fixed, and we have observed the effect with a variety of initiation-region promoter variants. The phenomenon is also observed during promoter-independent transcription when transcribing a homopolymeric template such as poly(dC). Under conditions where the active site of the RNAP cannot extend beyond the third nucleotide in the template strand and the maximum length of the RNA:DNA hybrid cannot exceed three base-pairs (i.e. when synthesizing oligoG products due to transcript slippage at a promoter that initiates with the sequence +1 GGG...) the mutant RNAP gives rise to a normal spectrum of products 2 to 14 nt in length with no evidence of a block at 5 nt. Neither promoter binding nor promoter melting appears to be involved in this phenotype, as the mutant RNAP binds normally to promoter sequences and the behavior of the enzyme is unaffected by removal of the non-template strand in the initiation region of the promoter or on a supercoiled template. Importantly, the mutant RNAP is defective in binding single strand oligomers of RNA. These results suggest that the affected region of the RNAP may form part of the RNA product binding site and may be involved in the transition from an unstable initiation complex to a stable elongation complex, perhaps by sensing the presence of a nascent RNA and/or RNA:DNA hybrid.

Amino Acids↗
PubMed1994

The exchange of the discriminator base A73 for G is alone sufficient to convert human tRNA(Leu) into a serine-acceptor in vitro.

Transfer RNA (tRNA) identify is maintained by the highly specific interaction of a few defined nucleotides or groups of nucleotides, called identity elements, with the cognate aminoacyl-tRNA synthetase, and by nonproductive interactions with the other 19 aminoacyl-tRNA synthetases. Most tRNAs have a set of identity elements in at least two locations, commonly in the anticodon loop or in the acceptor stem, and at the discriminator base position 73. We have used T7 RNA polymerase transcribed tRNAs to demonstrate that the sole replacement of the discriminator base A73 of human tRNA(Leu) with the tRNA(Ser)-specific G generates a complete identity switch to serine acceptance. The reverse experiment, the exchange of G73 in human tRNA(Ser) for the tRNA(Leu-specific A, causes a total loss of serine specificity without creating any leucine acceptance. These results suggest that the discriminator base A73 of human tRNA(Leu) alone protects this tRNA against serylation by seryl-tRNA synthetase. This is the first report of a complete identity switch caused by an exchange of the discriminator base alone.

Acylation↗
PubMed2001

Molecular basis for telomere repeat divergence in budding yeast.

Telomerase is a ribonucleoprotein enzyme that adds repetitive sequences to the ends of linear chromosomes, thereby counteracting nucleotide loss due to incomplete replication. A short region of the telomerase RNA subunit serves as template for nucleotide addition onto the telomere 3' end. Although Saccharomyces cerevisiae contains only one telomerase RNA gene, telomere repeat sequences are degenerate in this organism. Based on a detailed analysis of the telomere sequences specified by wild-type and mutant RNA templates in vivo, we show that the divergence of telomere repeats is due to abortive reverse transcription in the 3' and 5' regions of the template and due to the alignment of telomeres in multiple registers within the RNA template. Through the interpretation of wild-type telomere sequences, we identify nucleotides in the template that are not accessible for base pairing during substrate annealing. Rather, these positions become available as templates for reverse transcription only after alignment with adjacent nucleotides has occurred, indicating that a conformational change takes place upon substrate binding. We also infer that the central part of the template region is reverse transcribed processively. The inaccessibility of certain template positions for alignment and the processive polymerization of the central template portion may serve to reduce the possible repeat diversification and enhance the incorporation of binding sites for Rap1p, the telomere binding protein of budding yeast.

Base Sequence↗
PubMed1992

On the functional role of the Tyr-639 residue of bacteriophage T7 RNA polymerase.

Substitution of Asp for a Tyr residue normally present at position 639 of the bacteriophage T7 RNA polymerase leads to a drastic drop in the enzymatic activity. This mutation does not affect the enzyme-promoter interaction but decreases the ability of the RNA polymerase to discriminate between GTP and ATP molecules, resulting in a decrease in the rate of the incorporation of the nucleotide into the RNA chain.

Amino Acid Sequence↗
PubMed2002

The spectrum of spontaneous rifampin resistance mutations in the rpoB gene of Bacillus subtilis 168 spores differs from that of vegetative cells and resembles that of Mycobacterium tuberculosis.

Mutations causing rifampin resistance in vegetative cells of Bacillus subtilis 168 have thus far been mapped to a rather restricted set of alterations at either Q469 or H482 within cluster I of the rpoB gene encoding the beta subunit of RNA polymerase. In this study, we demonstrated that spores of B. subtilis 168 exhibit a spectrum of spontaneous rifampin resistance mutations distinct from that of vegetative cells. In addition to the rpoB mutations Q469K, Q469R, and H482Y previously characterized in vegetative cells, we isolated a new mutation of rpoB, H482R, from vegetative cells. Additional new rifampin resistance mutations arising from spores were detected at A478N and most frequently at S487L. The S487L change is the predominant change found in rpoB mutations sequenced from rifampin-resistant clinical isolates of Mycobacterium tuberculosis. The observations are discussed in terms of the underlying differences of the DNA environment within dormant cells and vegetatively growing cells.

Amino Acid Sequence↗
PubMed1994

Analysis of a rape case by direct sequencing of the human immunodeficiency virus type 1 pol and gag genes.

Transmission of human immunodeficiency virus type 1 (HIV-1) from a male accused of rape and deliberate transmission of HIV-1 was investigated by sequencing of the HIV-1 pol and gag genes from virus obtained from the male and from the female victim. Parts of the reverse transcriptase and p17gag genes were amplified and directly sequenced from uncultured peripheral blood mononuclear cells. The sequences were compared with sequences from 21 unrelated HIV-1-infected controls from the same geographic area (Stockholm, Sweden). Bootstrap analysis of phylogenetic trees demonstrated that the sequences from the female were significantly more closely related to the sequences from the male than to sequences from the controls. Furthermore, we found that the male and female shared two distinct genetic variants of HIV-1. In p17gag the major variant had an unusual, out-of-frame deletion of 3 nucleotides which the minor variant lacked. These results indicated that the male had transmitted more than one infectious unit to the female. From this study we concluded that it was highly likely that the HIV-1 strains carried by the male and female were closely epidemiologically linked.

Base Sequence↗
PubMed2004

T7 RNA polymerase transcription with 5-position modified UTP derivatives.

Seven UTP derivatives modified at the 5-position through an amide linkage were tested as substrates for T7 RNA polymerase (T7 RNAP) transcription. All UTP derivatives gave good yields of full-length transcript even from DNA templates that showed a significant number of abortive transcripts using unmodified UTP. A kinetic assay to determine the relative K(m) and V(max) for T7 RNAP transcription gave surprisingly similar values for UTP and the 5-position hydrophobic modifications phenyl, 4-pyridyl, 2-pyridyl, indolyl, and isobutyl. The 5-position modifications imidazole and amino, which could both be positively charged, gave K(m) values significantly higher than UTP. All seven UTP derivatives gave relative V(max) values similar to UTP, indicating that insertion of these modified bases into the transcript did not impede its elongation.

Bacteriophage T7↗
PubMed2002

[Mutations in beta'-subunit of the Escherichia coli RNA-polymerase influence interaction with downstream duplex DNA in the elongation complex].

RNA polymerase (RNAP) exhibits absolute processivity being capable of synthesizing RNA 10(3)-10(5) nucleotides in length without breaking contact with the DNA template. Stability of the elongation complex is thought to depend, in particular, on the RNAP-DNA interactions downstream along the run of transcription. We studied the effects of several deletions and insertions in the RNAP beta'-subunit N-terminal region, which presumably interacts with the downstream duplex DNA in the elongation complex. Most of the mutations obtained led to gross defects in RNAP assembly and disturbed catalytic activity of the enzyme. The mutations reduced stability of both promoter and elongation complexes, probably because they altered the contacts between RNAP and the downstream duplex DNA.

Amino Acid Sequence↗
PubMed1997

A novel retron that produces RNA-less msDNA in Escherichia coli using reverse transcriptase.

Bacterial retroelements, or retrons, use reverse transcriptase (RT) to produce a multicopy single-stranded DNA (msDNA) molecule that is covalently linked to RNA. In these studies we show that a retron from Escherichia coli 110, a clinical isolate, produces a novel RNA-less msDNA with a 5' phosphate residue. The msDNA is a 74-nucleotide single-stranded DNA molecule with a stable stem-loop structure without a mismatched base pair. Only the genes encoding msDNA (msd), msdRNA (msr), and RT (ret) are required to produce the msDNA molecule. The organization of these genes on the retron was similar to that of other elements producing branched msDNA-RNA. The conserved guanine, which is the branched residue in msDNA-RNA complexes and is essential for branch formation, is also present. Site-directed mutagenesis showed that this guanine is essential for the production of RNA-less msDNA. We postulate that the RNA-less msDNA in strain 110 is produced by nucleolytic cleavage of the branched msDNA-RNA compound.

Amino Acid Sequence↗
PubMed2000

Characterization of the initiation sites of both polarity strands of a viroid RNA reveals a motif conserved in sequence and structure.

Viroids replicate through a rolling-circle mechanism in which the infecting circular RNA and its complementary (-) strand are transcribed. The precise site at which transcription starts was investigated for the avocado sunblotch viroid (ASBVd), the type species of the family of viroids with hammerhead ribozymes. Linear ASBVd (+) and (-) RNAs begin with a UAAAA sequence that maps to similar A+U-rich terminal loops in their predicted quasi-rod-like secondary structures. The sequences around the initiation sites of ASBVd, which replicates and accumulates in the chloroplast, are similar to the promoters of a nuclear-encoded chloroplastic RNA polymerase (NEP), supporting the involvement of an NEP-like activity in ASBVd replication. Since RNA folding appears to be kinetically determined, the specific location of both ASBVd initiation sites provides a mechanistic insight into how the nascent ASBVd strands may fold in vivo. The approach used here, in vitro capping and RNase protection assays, may be useful for investigating the initiation sites of other small circular RNA replicons.

Base Sequence↗
PubMed2000

Sequence analysis of transposable elements in the sea squirt, Ciona intestinalis.

A systematic search of 1 Mb of genomic sequences from the sea squirt, Ciona intestinalis, revealed the presence of six families of transposable elements. The Cigr-1 retrotransposon contains identical 245-bp long terminal repeats (LTRs) and a 3,630-bp open reading frame (ORF) encoding translation products in the same order as the domains characteristic of gypsy/Ty3-type LTR retrotransposons. The closest homologs of the reverse transcriptase domain were in gypsy elements from Drosophila and the sushi element from the pufferfish. However, the capsid-nucleocapsid region shows the clearest homology to an echinoderm element, Tgr1. Database searches also indicated two classes of non-LTR retrotransposon, named Cili-1 and Cili-2. The Cili-1 sequences show matches to regions of the ORF2 product of mammalian L1 elements. The Cili-2 sequences possess similarity to the RNaseH domain of Lian-Aa1, a mosquito non-LTR retrotransposon. The most abundant element was a short interspersed nucleotide element named Cics-1 with a copy number estimated at 40,000. Cics-1 consists of two conserved domains separated by an A-rich stretch. The 172-bp 5' domain is related to tRNA sequences, whereas the 110-bp 3' domain is unique. Cics-1 is unusual, not just in its modular structure, but also in its lack of a 3' poly(A) tail or direct flanking repeats. A second abundant element, Cimi-1, has an A+T-rich 193-bp consensus sequence and 30-bp terminal inverted repeats (TIRs) and is usually flanked by A+T-rich 2-4-bp putative target site duplications-characteristics of miniature inverted-repeat transposable elements found in plants and insects. A single 2,444-bp foldback element was found, possessing long TIRs containing an A+T-rich internal domain, an array of subrepeats, and a flanking domain at the TIR ends; this is the first example of a chordate foldback element. This study provides the first systematic characterization of the families of transposable elements in a lower chordate.

Amino Acid Sequence↗