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M R Paule

Publications and source records attributed to M R Paule.

At least 37 records · Page 2Linked to original sources
PubMed1992

The start site of the Acanthamoeba castellanii ribosomal RNA transcription unit.

The 39S ribosomal RNA (rRNA) precursor has been isolated from Acanthamoeba castellanii. In vitro capping of the isolated RNA verified that it is the primary transcript and identified the 5' nucleotide as pppA. The position of the 5' coding nucleotide on the rRNA repeat unit sequence was identified using Northern blot, R-loop, and S1 nuclease mapping techniques. Dinucleotide priming of an in vitro transcription system stalled because of low initiating nucleotide concentration revealed that ApA maximally stimulates initiation of transcription. All of these results show that the underlined A in the sequence 5'-TATATATAAAGGGAC (RNA-like strand) coincides with the 5' nucleotide of the primary transcript. This identification is compatible with in vitro transcription experiments mapping the promoter for this transcription unit. The initiation sequences of rRNA genes from 14 species are compared, and a weak consensus for the initiator derived: [Formula; see text].

Acanthamoeba↗
PubMed1991

Sequence and organization of 5S RNA genes from the eukaryotic protist Acanthamoeba castellanii.

A 5S RNA genomic clone has been isolated from Acanthamoeba castellanii and the sequence of the coding region plus flanking DNA was determined. This clone encodes an RNA whose sequence matches that of 5S RNA from this organism. There is sequence similarity in the 5'-flanking region to other eukaryotic 5S RNA genes which require or are greatly affected by upstream regions for transcriptional activity. The immediate 3'-flanking region has a termination sequence similar to that found in all genes that are transcribed by RNA polymerase III. The 5S RNA genes of A. castellanii are dispersed, which is highly unusual, since the majority of eukaryotic organisms contain 5S genes clustered in tandem repeats. There may be up to 480 genes encoding 5S RNA in each A. castellanii cell.

Acanthamoeba↗
PubMed1991

Specific transcription of an Acanthamoeba castellanii 5S RNA gene in homologous nuclear extracts.

An RNA polymerase III in vitro transcription system has been developed from the protist Acanthamoeba castellanii. The system is dependent on a cloned 5S RNA gene and utilizes a nuclear extract which contains all the necessary protein components. The system is assembled from completely homologous components. Primer extension and RNA sequencing analysis confirm that the in vitro 5S RNA transcript is identical to the 5S RNA isolated from cells. The transcription complex forms unusually rapidly on the 5S RNA gene and is stable to challenge by excess competitor templates. Several 5' deletion mutants were constructed and indicate that the region upstream of -33 is dispensable. Deletion to +16 show the region between -33 and +16 to be required for transcription, a region outside the canonical internal control region.

Acanthamoeba↗
PubMed1988

Promoter occlusion during ribosomal RNA transcription.

The ability of RNA polymerase I to read through a promoter-bound transcription initiation factor (TIF) was assessed using a dimeric ribosomal RNA gene promoter. Transcription from the upstream promoter is unaffected by TIF bound to the downstream promoter; RNA polymerase I is freely able to read through DNA-bound TIF. In contrast, transcription from the downstream promoter is inhibited by the passage of RNA polymerase I. Readthrough by RNA polymerase I disrupts the downstream TIF-DNA complex, and alters the TIF footprint. A general function for sequences leading to transcription termination upstream of rRNA or other promoters may be the prevention of promoter occlusion in tandem arrays of genes.

Acanthamoeba↗
PubMed1988

Effects of single-base substitutions within the Acanthamoeba castellanii rRNA promoter on transcription and on binding of transcription initiation factor and RNA polymerase I.

Single-point mutations were introduced into the promoter region of the Acanthamoeba castellanii rRNA gene by chemical mutagen treatment of a single-stranded clone in vitro, followed by reverse transcription and cloning of the altered fragment. The promoter mutants were tested for transcription initiation factor (TIF) binding by a template commitment assay plus DNase I footprinting and for transcription by an in vitro runoff assay. Point mutations within the previously identified TIF interaction region (between -20 and -47, motifs A and B) indicated that TIF interacts most strongly with a sequence centered at -29 and less tightly with sequences upstream and downstream. Some alterations of the base sequence closer to the transcription start site (and outside the TIF-protected site) also significantly decreased specific RNA synthesis in vitro. These were within the region which is protected from DNase I digestion by polymerase I, but these mutations did not detectably affect the binding of polymerase to the promoter.

Acanthamoeba↗
PubMed1988

Events during eucaryotic rRNA transcription initiation and elongation: conversion from the closed to the open promoter complex requires nucleotide substrates.

Chemical footprinting and topological analysis were carried out on the Acanthamoeba castellanii rRNA transcription initiation factor (TIF) and RNA polymerase I complexes with DNA during transcription initiation and elongation. The results show that the binding of TIF and polymerase to the promoter does not alter the supercoiling of the DNA template and the template does not become sensitive to modification by diethylpyrocarbonate, which can identify melted DNA regions. Thus, in contrast to bacterial RNA polymerase, the eucaryotic RNA polymerase I-promoter complex is in a closed configuration preceding addition of nucleotides in vitro. Initiation and 3'-O-methyl CTP-limited translocation by RNA polymerase I results in separation of the polymerase-TIF footprints, leaving the TIF footprint unaltered. In contrast, initiation and translocation result in a significant change in the conformation of the polymerase-DNA complex, culminating in an unwound DNA region of at least 10 base pairs.

Acanthamoeba↗
PubMed1987

Eukaryotic RNA polymerase I promoter binding is directed by protein contacts with transcription initiation factor and is DNA sequence-independent.

RNA polymerase I binding to the eukaryotic ribosomal RNA gene promoter-transcription initiation factor (TIF) complex was examined by in vitro transcription and footprinting of a series of spacer mutants. Polymerase binds efficiently to the TIF-promoter complex independently of the DNA sequence in the polymerase interaction region and initiates transcription a fixed distance downstream of the TIF binding site on AT-rich templates. Methidiumpropyl-EDTA.FE(II) footprinting confirms minimal contacts between polymerase and DNA. We infer that polymerase is directed to the promoter by a DNA sequence-independent mechanism, solely by protein-protein contacts with TIF. An initiation step subsequent to binding requires special sequence characteristics in the transcription start site region.

Amoeba↗
PubMed1986

Regulation of eukaryotic ribosomal RNA transcription by RNA polymerase modification.

Forms of RNA polymerase I prepared from growing or encysted Acanthamoeba are equal in the ability to transcribe poly(dl:dC). Polymerase from cysts, whose rRNA genes are transcriptionally inactive, is unable to utilize the rDNA promoter in vitro, whereas the transcription initiation factor from cysts is fully able to bind the promoter and direct transcription. Footprinting shows that polymerase from cysts is functionally inactive because of its inability to bind to the promoter. The polymerase footprint moves downstream the appropriate number of base pairs upon various nucleotide additions, without affecting the factor footprint. These results support our hypothesis that rRNA synthesis in eukaryotes is regulated by polymerase I modification and not by alterations to additional DNA-binding proteins.

Amoeba↗
PubMed1985

The ribosomal RNA promoter of Acanthamoeba castellanii determined by transcription in a cell-free system.

The DNA sequences required for faithful initiation of ribosomal RNA transcription were determined. BAL-31 digestion was used to modify the rDNA template by introducing deletions from its 3'- and 5'-ends. The resulting mutant DNAs were tested for template activity individually or in competition with wild type utilizing an in vitro transcription system from Acanthamoeba castellanii. The results identify the sequence extending from -31 to +8 to be absolutely required for transcription. In addition; when the region between -47 and -32 is left intact, transcription is augmented.

Amoeba↗
PubMed1985

Footprinting of ribosomal RNA genes by transcription initiation factor and RNA polymerase I.

The binding of a species-specific transcription initiation factor (TIF) and purified RNA polymerase I to the promoter region of the 39S ribosomal RNA gene from Acanthamoeba were studied by using DNase I "footprinting." Conditions were chosen such that the footprints obtained could be correlated with the transcriptional activity of the TIF-containing fractions used and that the labeled DNA present would itself serve as a template for transcription. The transcription factor binds upstream from the transcription start site, protecting a region extending from around -14 to -67 on the coding strand, and -12 to -69 on the noncoding strand. The protein that binds to DNA within this region can be competed out by using wild-type promoters but not by using mutants which do not stably bind the factor. RNA polymerase I can form a stable complex in the presence of DNA and transcription factor, allowing footprinting of the complete transcription initiation complex. RNA polymerase I extends the protected region obtained with TIF alone to around +18 on the coding strand, and to +20 on the noncoding strand. This region is not protected by polymerase I in the absence of TIF. The close apposition of the regions protected by TIF and polymerase provides evidence that accurate transcription of the ribosomal gene may be achieved through protein-protein contacts as well as through DNA-protein interactions.

Amoeba↗
PubMed1985

Ribosomal RNA transcription: proteins and DNA sequences involved in preinitiation complex formation.

An in vitro transcription system consisting of partially purified transcription initiation factor(s) and purified RNA polymerase I from Acanthamoeba castellanii was used to study the mechanism of faithful initiation of ribosomal RNA transcription. Formation of a preinitiation complex between one or several auxiliary transcription proteins and the DNA template in the absence of RNA polymerase I was demonstrated. A series of 3'- and 5'-deletion mutants of the template was used in prebinding competition experiments and provided evidence for three distinct functional regions of the promoter: core motif A interacts with the transcription initiation factor(s) and is required for faithful transcription; the start motif is required for transcription, but it can be deleted without affecting the binding of transcription initiation factor(s); and motif B stabilizes preinitiation complex formation (in addition to core motif A), but it is dispensable for faithful initiation of transcription.

Amoeba↗
PubMed1984

In vitro evidence that eukaryotic ribosomal RNA transcription is regulated by modification of RNA polymerase I.

We have utilized a cell-free transcription system from Acanthamoeba castellanii to test the functional activity of RNA polymerase I and transcription initiation factor I (TIF-I) during developmental down regulation of rRNA transcription. The results strongly suggest that rRNA transcription is regulated by modification, probably covalent, of RNA polymerase I: (1) The level of activity of TIF-I in extracts from transcriptionally active and inactive cells is constant. (2) The number of RNA polymerase I molecules in transcriptionally active and inactive cells is also constant. (3) In contrast, though the specific activity of polymerase I on damaged templates remains constant, both crude and purified polymerase I from inactive cells have lost the ability to participate in faithful initiation of rRNA transcription. (4) Polymerase I purified from transcriptionally active cells has the same subunit architecture as enzyme from inactive cells. However, the latter is heat denatured 5 times faster than the active polymerase.

Amoeba↗
PubMed1984

Faithful initiation of ribosomal RNA transcription from cloned DNA by purified RNA polymerase I.

A faithful transcription system for ribosomal RNA genes has been developed by using components from the small free-living amoeba Acanthamoeba castellanii. The system utilizes protein-free recombinant DNA as a template and in addition requires a crude cell-free extract containing RNA polymerase I and a transcription initiation factor (TIF-I). The transcript is initiated at the same position as the in vivo precursor ribosomal RNA: templates truncated at various sites downstream of the transcription start site give rise to only the predicted runoff RNA transcripts, and the runoff transcript produced has a 5'-terminus identical with the 5'-terminus of the isolated ribosomal RNA precursor. Faithful initiation can be elicited by the DNA sequence extending from -55 to +19 in the template. Subclones containing this sequence yield only the predicted runoff RNAs regardless of the orientation of this fragment in the cloning vector DNA; thus, only the in vivo sense strand of the template is specifically transcribed in the in vitro system. The system is specific for the RNA polymerase responsible for the transcription of ribosomal RNA genes in vivo. Faithful transcription, like RNA polymerase I from Acanthamoeba, is insensitive to alpha-amanitin inhibition, and transcription is greatly stimulated by highly purified RNA polymerase I but not by RNA polymerases II or III. Conditions for optimal transcription were determined.

Amoeba↗
PubMed1983

Regulation of ribosomal RNA transcription during differentiation of Acanthamoeba castellanii: a review.

During the cellular differentiation induced by starvation of Acanthamoeba castellanii, the expression of a number of genes is regulated. Evidence is reviewed that at least one of these, the precursor ribosomal RNA transcription unit, is regulated at the level of transcription. The structure of the rRNA transcription unit and of the RNA polymerases responsible for transcription in Acanthamoeba are reviewed. Utilizing an in vitro transcription system constructed from these components, preliminary evidence has been obtained that pre-rRNA gene expression is regulated by a modification of RNA polymerase I that affects the enzyme's ability to participate efficiently in the initiation of transcription. These results are reviewed in relation to other known mechanisms of transcriptional regulation in eukaryotes.

Amoeba↗
PubMed1981

Ribosomal ribonucleic acid repeat unit of Acanthamoeba castellanii: cloning and restriction endonuclease map.

The repeat unit coding for the precursor to 18S, 5.8S, and 26S ribosomal ribonucleic acids (rRNAs) has been cloned from the free-living soil amoeba Acanthamoeba castellanii. The cloned deoxyribonucleic acid (DNA) was mapped with 11 restriction endonucleases and by R-loop mapping. The entire repeat unit is 12 kbp (kilobase pairs) in length and contains sites for EcoRI, SmaI, BglII, SstI, Bam-HI, PstI, KpnI, HindIII, and XbaI but not for XhoI or SalI. All of the repeat units in the nuclear DNA appear to be identical, and no introns were detected. However, the regions which code for the two RNAs which comprise the 26S RNA are separated by a gap of approximately 200 base pairs. Unlike some other lower eukaryotes, the 5S RNA gene is not linked to this repeat unit. A fragment of the repeat unit which contains the initiation sequence of the putative precursor has been subcloned into pBR322 for use in vitro transcription studies.

Amoeba↗
PubMed1979

DNA-dependent RNA polymerases from Acanthamoeba castellanii. Comparative subunit structures of the homogeneous enzymes.

The constituent polypeptides of the three classes of DNA-dependent RNA polymerase from Acanthamoeba castellanii were compared by several electrophoretic methods. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) reveals that a number of polypeptide components of the isozymes have identical molecular weights. Two-dimensional electrophoresis (isoelectric focusing in 8 M urea:SDS-polyacrylamide gel electrophoresis) demonstrates that the polypeptides of identical molecular weights also have identical isoelectric pH values. These polypeptides were also coincident after electrophoresis in 8 M urea at acidic or basic pH values followed by a second electrophoretic separation in the presence of SDS. By these criteria, subunits of molecular weight 13,300, 15,500, 17,500, 22,500, 37,000, and 39,000 are indistinguishable in polymerase I and III. The 13,300, 15,500, and 22,500 subunits are also shared by the class II polymerase. In addition, electrophoresis in 8 M urea under basic conditions reveals microheterogeneity in the 17,500 molecular weight subunit. The strikingly similar pattern of common subunits between yeast and Acanthamoeba suggests that a universal arrangement of functional units may be an essential feature of the eukaryotic polymerases.

Amoeba↗