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A P Wolffe

Publications and source records attributed to A P Wolffe.

At least 19 recordsLinked to original sources
PubMed1992

Influence of chromatin folding on transcription initiation and elongation by RNA polymerase III.

Nucleosomes were assembled onto either closed circular plasmids containing a single Xenopus 5S RNA gene or a linear tandemly repeated array of Lytechinus 5S RNA genes. Both chromatin templates were found to vary in their extent of compaction, depending upon the type and concentration of cation in solution. Compaction of these chromatin templates led to a significant inhibition of both transcription initiation and elongation by RNA polymerase III. Thus, the transcriptional repression observed after incorporation of genes into chromatin depends not only on occlusion of the promoter elements through direct contact with histones but also on compaction of nucleosomal arrays which occurs under the conditions of the transcription reactions.

Animals↗
PubMed1992

Histones H2A/H2B inhibit the interaction of transcription factor IIIA with the Xenopus borealis somatic 5S RNA gene in a nucleosome.

A Xenopus borealis somatic 5S RNA gene was assembled with either the complete octamer of histones, (H2A/H2B/H3/H4)2, or the (H3/H4)2 tetramer of histones that comprises the central protein kernel of the nucleosome. Gel-mobility shifts, DNase I protection, and immunoblotting assays demonstrate that the class III transcription factor IIIA (TFIIIA) readily interacts with 5S DNA associated with the tetramer but that little or no binding is detected when 5S DNA is associated with the full octamer of histones. Thus, the presence of histones H2A and H2B in the 5S nucleosome significantly inhibits the interaction of TFIIIA with its cognate binding site within the 5S RNA gene. We propose that either the depletion of histones H2A and H2B from preexisting nucleosomes or the staged assembly of chromatin after replication in which a tetramer of histones H3/H4 associates with DNA before histones H2A/H2B will facilitate the binding of transcription factors to their cognate DNA sequences.

Animals↗
PubMed1992

The interaction of transcription factors with nucleosomal DNA.

Nucleosome positioning is proposed to have an essential role in facilitating the regulated transcription of eukaryotic genes. Some transcription factors can bind to DNA when it is appropriately wrapped around the histone core, others cannot bind due to the severe deformation of DNA structure. The staged assembly of nucleosomes and positioning of histone-DNA contacts away from promoter elements can facilitate the access of transcription factors to DNA. Positioned nucleosomes can also facilitate transcription through providing the appropriate scaffolding to bring regulatory factors bound at dispersed sites into juxtaposition.

Animals↗
PubMed1992

In vitro transcription by RNA polymerase II in extracts of Xenopus oocytes, eggs, and somatic cells.

We describe procedures for preparing extracts of Xenopus oocytes, eggs, and somatic cells that will accurately transcribe class II genes. A variety of viral and Xenopus promoters direct the accurate initiation of transcription by RNA polymerase II in these extracts. Optimal ionic conditions (100-200 mM KCl, 12 mM MgCl2), template concentration (20-40 micrograms/ml), incubation time (30-60 min), and temperature (25 degrees C) for class II gene transcription are described.

Animals↗
PubMed1992

Characterization of RNA polymerase II-dependent transcription in Xenopus extracts.

We examine the RNA polymerase II-dependent transcription directed by several promoters in extracts prepared from distinct developmental stages of Xenopus laevis. RNA polymerase II accurately initiates transcription from the cytomegalovirus, herpes simplex virus thymidine kinase, and Xenopus heat-shock protein (hsp) 70 promoters. The efficiency of transcription of these different promoters is dependent on whether extracts from oocytes, eggs, or somatic cells are used and on the temperature of incubation. In contrast to the viral promoters, the hsp 70 promoter is more active at heat shock temperatures in oocyte and egg extracts (31 degrees-34 degrees C) than at physiological temperatures for Xenopus (20 degrees-25 degrees C). These in vitro transcription extracts should be useful in examining the molecular mechanisms responsible for differential gene expression during Xenopus development.

Animals↗
PubMed1992

New insights into chromatin function in transcriptional control.

Transcription requires the recognition of numerous DNA sequences by diverse transcription factors, which together assemble large nucleoprotein complexes that tether RNA polymerase and facilitate the initiation of RNA synthesis. In vivo the assembly of these transcription complexes occurs in a nuclear environment where the template DNA is compacted more than 10(5)-fold through the assembly of chromatin. Our current knowledge of chromatin structure raises the major issues of the accessibility of DNA to both transcription factors and RNA polymerase and of the progression of RNA polymerase along the chromatin fiber. Novel genetic and biochemical approaches have established that specific chromatin structures have a major impact on the transcription process. Precise alterations in histone sequence, nucleosome structure, and folding of the chromatin fiber influence both the activation and repression of genes. These results offer substantial insight into the role of modulations in chromosome structure during vertebrate development. Transcription factors, histones, and the organization of DNA within the chromosome are intimately linked, and only when the correct framework is assembled can they function together to achieve correct genetic regulation.

Animals↗
PubMed1992

DNA gyrase, CS7.4, and the cold shock response in Escherichia coli.

We identify the A subunit of DNA gyrase as a cold shock protein whose synthesis is sustained following transfer of exponentially growing cultures of Escherichia coli from 37 to 10 degrees C. After a lag period in which its synthetic rate declines, synthesis of the A subunit of DNA gyrase increases relative to that of total protein. The duration of the lag period in synthesis and the synthetic rate of the A subunit appear dependent on the synthesis of a rapidly induced cold shock protein, CS7.4. The promoter of the gyrA gene contains specific binding sites for the CS7.4 protein, suggesting that CS7.4 acts at the transcriptional level to facilitate continued A-subunit synthesis. As synthesis of the B subunit of DNA gyrase is also sustained during cold shock, we suggest that an increase in the amount of DNA gyrase per cell might occur, which would potentially adapt the cells for growth at reduced temperatures (10 degrees C).

Bacterial Proteins↗
PubMed1992

Chromosomal organization of Xenopus laevis oocyte and somatic 5S rRNA genes in vivo.

We describe the chromosomal organization of the major oocyte and somatic 5S RNA genes of Xenopus laevis in chromatin isolated from erythrocyte nuclei. Both major oocyte and somatic 5S DNA repeats are associated with nucleosomes; however, differences exist in the organization of chromatin over the oocyte and somatic 5S RNA genes. The repressed oocyte 5S RNA gene is protected from nuclease digestion by incorporation into a nucleosome, and the entire oocyte 5S DNA repeat is assembled into a loosely positioned array of nucleosomes. In contrast, the potentially active somatic 5S RNA gene is accessible to nuclease digestion, and the majority of somatic 5S RNA genes appear not to be incorporated into positioned nucleosomes. Evidence is presented supporting the stable association of transcription factors with the somatic 5S RNA genes. Histone H1 is shown to have a role both in determining the organization of nucleosomes over the oocyte 5S DNA repeat and in repressing transcription of the oocyte 5S RNA genes.

Animals↗
PubMed1992

The RNA polymerase III terminator used by a B1-Alu element can modulate 3' processing of the intermediate RNA product.

The dispersion of short interspersed elements (SINEs) probably occurred through an RNA intermediate. B1 is a murine homolog of the human SINE Alu; these elements are composed of 5' G + C-rich regions juxtaposed to A-rich tracts and are flanked by direct repeats. Internal promoters direct RNA polymerase III to transcribe B1 and Alu elements and proceed into the 3' flanking DNA until it reaches a (dT)4 termination signal. The resulting transcripts contain 3'-terminal oligo(U) tracts which can presumably base pair with the A-rich tract to form self-primed templates for reverse transcriptase and retrotransposition. Nuclear extracts from mouse tissue culture cells contain an RNA processing activity that removes the A-rich and 3'-terminal regions from purified B1 RNAs (R. Maraia, Nucleic Acids Res. 19:5695-5702, 1991). In this study, we examined transcription and RNA processing in these nuclear extracts. In contrast to results with use of purified RNA, nascent transcripts synthesized in nuclear extract by RNA polymerase III are not processed, suggesting that the transposition-intermediate-like RNA is shielded from processing by a protein(s). Alteration of an AATTTT TAA termination signal to a GCTTTTGC signal activated processing by greater than 100-fold in coupled transcription/processing reactions. A similar difference was found when expression was compared in frog oocytes. No difference in processing was found if the transcripts were made by T7 RNA polymerase in the presence of the nuclear extract, indicating that the different processing effects of the two terminators were dependent on synthesis by polymerase III. The modulation of processing of B1-Alu transcripts and the potential for retrotransposition of B1 and Alu DNA sequences are discussed.

Animals↗
PubMed1992

DNA binding, multimerization, and transcription stimulation by the Xenopus Y box proteins in vitro.

The Y box factors bind to a specific DNA sequence (the Y box, containing a reverse CCAAT element) and have been implicated in the regulation of transcription. We have used deletion mutagenesis to define the protein domains of two Xenopus Y box factors, FRG Y1 and FRG Y2, that are essential for DNA binding, multimerization, and transcription. A domain of the Y box factors homologous to an Escherichia coli cold shock protein is required for DNA binding. Both the E. coli protein and the Y box factors recognize DNA sequences with similar selectivity. The conserved region between these proteins does not contain any previously defined DNA-binding motifs. The hydrophilic C-terminal tail of the proteins contributes to the assembly of nucleoprotein complexes. This region contains an unusual pattern of basic and acidic amino acids and represents a new type of domain mediating protein-protein interactions in transcription factors. Both the DNA-binding and the multimerization domains are important for stimulating transcription from the Xenopus hsp70 promoter in vitro.

Amino Acid Sequence↗
PubMed1991

The histone core exerts a dominant constraint on the structure of DNA in a nucleosome.

We have examined the structures of unique sequence, A/T-rich DNAs that are predicted to be relatively rigid [oligo(dA).oligo(dT)], flexible [oligo[d(A-T)]], and curved, using the hydroxyl radical as a cleavage reagent. A 50-base-pair segment containing each of these distinct DNA sequences was placed adjacent to the T7 RNA polymerase promoter, a sequence that will strongly position nucleosomes. The final length of the DNA fragments was 142 bp, enough DNA to assemble a single nucleosome. Cleavage of DNA in solution, while bound to a calcium phosphate crystal, and after incorporation into a nucleosome is examined. We find that the distinct A/T-rich DNAs have very different structural features in solution and helical periodicities when bound to a calcium phosphate. In contrast, the organization of the different DNA sequences when associated with a histone octamer is very similar. We conclude that the histone core exerts a dominant constraint on the structure of DNA in a nucleosome and that inclusion of these various unique sequences has only a very small effect on overall nucleosome stability and structure.

Animals↗
PubMed1991

Histone contributions to the structure of DNA in the nucleosome.

We describe the application of the hydroxyl radical footprinting technique to examine the contribution of the core histone tails and of histones H3 and H4 to the structure of DNA in the nucleosome. We first establish that, as was previously determined for a nucleosome containing a unique sequence of DNA, mixed-sequence nucleosomes contain two distinct regions of DNA structure. The central three turns of DNA in the nucleosome have a helical periodicity of approximately 10.7 base pairs per turn, while flanking regions have a periodicity of approximately 10.0 base pairs per turn. Removal of the histone tails does not change the hydroxyl radical cleavage pattern in either mixed- or unique-sequence nucleosome samples. A tetramer of histones H3 and H4, (H3/H4)2, organizes the central 120 base pairs of DNA identically to that found in the nucleosome. Moreover, "tailless" octamers and the (H3/H4)2 tetramer recognize the same nucleosome positioning signals as the intact octamer.

Animals↗
PubMed1991

Superhelical stress and nucleosome-mediated repression of 5S RNA gene transcription in vitro.

Nucleosomes were assembled on a plasmid carrying a Xenopus somatic 5S RNA gene prepared at different superhelix densities. The gene was preferentially assembled into a positioned nucleosome which was stable to superhelical stress. No evidence for a conformational change in the nucleosome was found, even under extreme negative superhelical stress. Transcription in an extract from Xenopus oocyte nuclei was repressed to a degree which depended on the number of nucleosomes assembled. Topoisomerase activity in the extract was effectively inhibited by camptothecin, which had no effect on transcription. Transcription of reconstitutes remained repressed relative to naked plasmids, and was independent of superhelix density. Transcripts from reconstitutes were derived solely from nucleosome-free genes. Thus, a histone octamer positioned on the gene was sufficient to block its transcription. Tryptic removal of the core histone tail domains had no effect on transcription at any superhelix density. Transcription of reconstitutes containing H3/H4 tetramers was also repressed, but not eliminated (unlike reconstitutes containing octamers), and repression was independent of superhelix density. We suggest that removal of histones H2A/H2B from the nucleosome facilitates activation of transcription in the extract. We conclude that superhelical stress alone does not activate transcription of a 5S RNA gene assembled into a nucleosome in vitro.

Animals↗
PubMed1991

RNA polymerase III transcription.

Remarkable progress has been made in defining the functional significance of the protein-DNA interactions involved in transcription complex formation on yeast tRNA and 5S RNA genes. This new information leads to a re-evaluation of how the class III gene transcription machinery operates.

Animals↗
PubMed1991

Transcription complex disruption caused by a transition in chromatin structure.

Chromatin structure is known to influence class III gene expression in vitro. We describe the active transcription of Xenopus class III genes following replication and assembly into chromatin by using Xenopus egg extracts. Changes in the structure of this active chromatin dependent on the presence of exogeneous Mg2+ ATP or on the addition of a mixture of histones H2A and H2B are shown to lead to the selective repression of Xenopus 5S RNA genes. Preexisting transcription complexes on 5S DNA are disrupted following the reorganization of a "disordered" histone-DNA complex into a structure consisting of physiologically spaced nucleosomes. Thus, we demonstrate that chromatin structural transitions can have dominant and specific effects on transcription.

Adenosine Triphosphate↗