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J M Gottesfeld

Publications and source records attributed to J M Gottesfeld.

At least 55 records · Page 3Linked to original sources
PubMed1990

Additional intragenic promoter elements of the Xenopus 5S RNA genes upstream from the TFIIIA-binding site.

The major promoter element of the Xenopus laevis 5S RNA gene is located within the transcribed region of the gene and forms the binding site for the transcription initiation factor TFIIIA. We report an analysis of deletion and substitution mutations within the coding region of the major oocyte-type 5S gene of X. laevis. Our results differ from those of previous mutagenesis studies conducted on the somatic-type genes of Xenopus borealis and X. laevis. Transcription assays in whole oocyte S-150 extracts, with both oocyte- and somatic-type mutants, revealed additional promoter elements between the start site for transcription and the binding site for TFIIIA. These sequences regulate the efficiency of binding TFIIIC, a transcription factor required by the genes transcribed by RNA polymerase III containing intragenic promoters. Under TFIIIC-limiting conditions, the somatic-type gene had a 10-fold-higher affinity for TFIIIC than did the major oocyte-type 5S gene. One mutation in the oocyte-type gene (nucleotides +33 to +39) reduced TFIIIC affinity and transcriptional activity four- to fivefold. Differences in TFIIIC affinity between oocyte- and somatic-type genes may contribute to the differential transcription of these genes observed during Xenopus embryogenesis.

Animals↗
PubMed1989

Transcription factor IIIA induced bending of the Xenopus somatic 5S gene promoter.

Transcription factor IIIA (TFIIIA), the canonical zinc-finger protein, is a protein of relative molecular mass 39,000 (39K) that is required for transcription of 5S-ribosomal subunit genes in Xenopus. It binds in a sequence-specific manner to the internal control region of the 5S gene (see Fig. 1) and facilitates transcription of the gene by RNA polymerase III. It also binds to the 5S gene product to form a 7S ribonucleoprotein particle. In oocytes the 7S particle acts as a storage form of the RNA to be utilized later in development. TFIIIA binds to DNA through its 30 K N-terminal domain, which contains nine zinc-fingers. TFIIIA was the first protein described to have this type of DNA binding motif, but numerous other proteins have now been shown to have zinc-finger domains. A structure for a single zinc-finger from the yeast protein ADR1, was recently proposed based on two-dimensional NMR data (ref. 8), and a similar structure was proposed based on comparison with crystal structures of other metalloproteins. Although models for the interaction of TFIIIA with the 5S-ribosomal gene DNA have been proposed, based on nuclease digestion and methylation interference data, little precise structural information is available for TFIIIA and the physical basis for the interaction of zinc-fingers with DNA is not understood. Using both circular permutation and circularization assays we provide convincing biochemical evidence that TFIIIA bends the DNA at the internal promoter of the 5S gene.

Animals↗
PubMed1989

Pathways of nucleoprotein assembly on 5S RNA genes in a Xenopus oocyte S-150 extract.

Conditions for transcription and nucleosome assembly of plasmids bearing Xenopus 5S RNA genes have been monitored in the whole oocyte S-150 extract (1). We find that the optimal conditions for transcription differ substantially from optimal conditions for nucleosome assembly. DNA molecules bearing as few as 50% of the native density of nucleosomes are transcriptionally inert. Although the 5S gene-specific transcription factor TFIIIA is in excess in this extract, these nucleosome reconstitutes do not exhibit TFIIA-like DNase footprints nor do these reconstitutes bind exogenous TFIIIA. We have also examined the nucleotide requirement for DNA supercoiling and for generation of 5S gene transcription complexes. Supercoiling associated with nucleosome assembly does not require ATP; however, nucleotide hydrolysis is required for establishment of active complexes. Phosphorylation of a 200 kdalton protein occurs in a 5S DNA-dependent manner concurrent with the generation of primed transcription complexes. Results of nondenaturing gel electrophoresis coupled with a second dimension of SDS gel electrophoresis suggest that the 200 kD protein may be a component of the 5S RNA gene transcription complex.

Adenosine Triphosphate↗
PubMed1988

Xenopus transcription factor IIIA forms a complex of covalent character with 5S DNA.

The 5S gene-specific transcription factor TFIIIA forms an exceptionally stable complex with the internal promoter of the 5S RNA gene. Approximately 1 to 5% of TFIIIA-DNA or deoxyoligonucleotide complexes are stable to harsh denaturation conditions and can be resolved by electrophoresis in the presence of SDS. These complexes are resistant to acidic conditions (0.1 N HCl, 55 degrees C, 2h) suggesting that the interaction may be through a covalent bond. Complex formation does not result in DNA strand scission and studies of the chemical sensitivity of the complex suggest that the TFIIIA-DNA linkage may be through a phosphoramidate bond. Covalent complexes are formed with both the noncoding (RNA-like) and coding strands of the internal control region. The predominant sites of TFIIIA-DNA adducts have been mapped to the 3' end of the 5S gene internal control region, the region previously shown to exhibit essential guanine and phosphate contacts with TFIIIA.

Animals↗
PubMed1987

Differential transcription of Xenopus oocyte and somatic-type 5 S genes in a Xenopus oocyte extract.

We have measured the transcription of Xenopus laevis oocyte and somatic-type 5 S RNA genes in S-150 extracts prepared from ovaries of mature X. laevis females (Glikin, G. C., Ruberti, I., and Worcel, A. (1984) Cell 37, 33-41). We find that somatic-type 5 S genes are transcribed at least 40-fold more efficiently than oocyte-type 5 S genes. Since adenovirus VA, Xenopus OAX, and Xenopus tRNAMet genes are all transcribed at levels similar to that of the somatic-type 5 S gene, this difference reflects a low level of oocyte-type 5 S gene transcription. Somatic-type 5 S transcription is a linear function of somatic-type 5 S DNA concentration and this, together with the efficient transcription of other class III genes, suggests that RNA polymerase III and the general class III transcription factors are not limiting under the conditions used here. Moreover, the 5 S gene-specific transcription factor TFIIIA does not limit transcription under these conditions as preincubation with purified TFIIIA does not alter transcription of either gene, and both genes exhibit characteristic TFIIIA footprints under transcription conditions in the S-150. Somatic-type 5 S DNA incubated in the S-150 and then injected into oocyte nuclei is actively transcribed whereas oocyte-type 5 S DNA treated in the same manner is inactive. We conclude that factors in the S-150 distinguish between somatic and oocyte-type 5 S genes, assembling active complexes preferentially on the former and inactive complexes preferentially on the latter. This process is not explained by binding properties of transcription factors for these genes as currently understood and represents a previously unrecognized mechanism of transcriptional regulation of the Xenopus 5 S genes.

Animals↗
PubMed1987

Transcriptionally inactive oocyte-type 5S RNA genes of Xenopus laevis are complexed with TFIIIA in vitro.

An extract from whole oocytes of Xenopus laevis was shown to transcribe somatic-type 5S RNA genes approximately 100-fold more efficiently than oocyte-type 5S RNA genes. This preference was at least 10-fold greater than the preference seen upon microinjection of 5S RNA genes into oocyte nuclei or upon in vitro transcription in an oocyte nuclear extract. The approximately 100-fold transcriptional bias in favor of the somatic-type 5S RNA genes observed in vitro in the whole oocyte extract was similar to the transcriptional bias observed in developing Xenopus embryos. We also showed that in the whole oocyte extract, a promoter-binding protein required for 5S RNA gene transcription, TFIIIA, was bound both to the actively transcribed somatic-type 5S RNA gene and to the largely inactive oocyte-type 5S RNA genes. These findings suggest that the mechanism for the differential expression of 5S RNA genes during Xenopus development does not involve differential binding of TFIIIA to 5S RNA genes.

Animals↗
PubMed1987

DNA sequence-directed nucleosome reconstitution on 5S RNA genes of Xenopus laevis.

Nucleosomes were reconstituted in vitro with several singly end-labeled restriction fragments derived from a cloned somatic-type 5S RNA gene of Xenopus laevis and purified nucleosome core particles from Xenopus cultured cells or chicken erythrocytes. Nucleosome locations were determined by digestion of the reconstitutes with exonuclease III and DNase I and were the same for all fragments investigated, extending from 20 base pairs (bp) within the 5S gene to 80 bp beyond the 3' end of the gene. Both core particles and crude nuclear extracts gave equivalent results, suggesting that no factors other than the core histones are responsible for recognition of DNA sequence during reconstitution. The histone octamer and the 5S gene-specific transcription factor TFIIIA both bind to the same region and face of 5S DNA, and nucleosome reconstitution on the 5S gene excluded binding of TFIIIA. The helical repeat of somatic-type 5S DNA in solution was measured by the band shift method and was 10.5 to 10.6 bp per turn over the region of the TFIIIA-binding site. The difference in helical repeat between DNA in solution and on the surface of the nucleosome (10.0-bp spacing between DNase I cutting sites) may explain the linking number paradox.

Animals↗
PubMed1986

Novobiocin inhibits RNA polymerase III transcription in vitro by a mechanism distinct from DNA topoisomerase II.

The role of DNA topoisomerases in eucaryotic class III gene transcription in vitro has been studied through the use of inhibitory drugs and antisera to DNA topoisomerases I and II. The DNA topoisomerase II inhibitors, novobiocin and coumermycin AI, were found to inhibit transcription of cloned 5S and tRNA genes. Novobiocin acts by interfering with an ATP-requiring step in the pathway to stable preinitiation complex formation. However, it is unlikely that this step reflects the enzymatic action of DNA topoisomerase II since a specific inhibitor of this enzyme (VM-26) and anti-DNA topoisomerase II antibodies fail to inhibit transcription under conditions where topoisomerase II enzymatic activity is inhibited. Similarly, a specific inhibitor of DNA topoisomerase I (camptothecin) and anti-DNA topoisomerase I antibodies fail to inhibit class III gene transcription. These results argue against a role for either DNA topoisomerase in 5S or tRNA gene transcription in vitro.

Aminocoumarins↗
PubMed1985

Torsional stress induces an S1 nuclease-hypersensitive site within the promoter of the Xenopus laevis oocyte-type 5S RNA gene.

The internal promoter of the Xenopus laevis oocyte-type 5S RNA gene is preferentially cleaved by S1 and Bal-31 nucleases in plasmid DNA. S1 nuclease sensitivity is largely dependent on supercoiling; however, Bal-31 cleaves within the 5S RNA gene in linear as well as in supercoiled DNA. The S1 nuclease-hypersensitive site is centered at position +48-52 of the gene at the 5' boundary of the promoter. A DNAase I-hypersensitive site is induced at this position upon binding of the transcription factor, TFIIIA, specific for the 5S RNA gene. The somatic-type 5S RNA gene promoter is not preferentially cleaved by S1 nuclease or Bal-31 nuclease in supercoiled DNA, nor does TFIIIA induce a DNase I site at position +50. This differential promoter response may be related to a 4-fold difference in TFIIIA affinity between the oocyte and somatic 5S RNA genes.

Animals↗
PubMed1984

Competition between Xenopus satellite I sequences and Pol III genes for stable transcription complex formation.

We have constructed hybrid plasmids bearing both Xenopus 5S RNA genes and satellite I sequences in order to test the effect of satellite DNA on 5S gene transcription. Satellite sequences inactivate 5S transcription in both HeLa S100 and Xenopus oocyte microinjection transcription assays. Inactivation of 5S transcription by satellite DNA is observed both in cis and in trans. Transcription of a tRNA gene is also precluded by satellite I DNA. The Xenopus satellite I repeat contains an RNA polymerase III transcription unit which is highly active in both assay systems. This promoter element is 10- to 25-fold more efficient than the 5S gene in transcription competition assays. This quantitative difference in affinity for transcription components may explain the inactivation of 5S transcription by satellite sequences. The satellite I promoter forms stable transcription complexes in vitro which do not dissociate for at least 30 rounds of transcription. Although stable complex formation on the satellite promoter is largely temperature independent over the range of 0-20 degrees, complex formation on both 5S and tRNA genes exhibits a steep temperature dependence characteristic of DNA helix unwinding. The DNA sequence requirements for stable complex formation on 5S genes have been determined using 5' deletion mutants.

Animals↗
PubMed1984

Control of neuronal gene expression.

Some 30,000 genes are expressed exclusively in the rat brain, many of which contain a genetic element called an identifier sequence located in at least one of their introns. The identifier sequences are transcribed by RNA polymerase III exclusively in neurons to produce two RNA species, BC1 and BC2, of 160 and 100 to 110 nucleotides. This transcriptional event may define regions of chromatin that contain neuronal-specific genes and may poise these genes for transcription by polymerase II by rendering the gene promoters accessible to soluble trans-acting molecules.

Animals↗
PubMed1984

Association of an RNA polymerase III transcription factor with a ribonucleoprotein complex recognized by autoimmune sera.

RNA polymerase III transcription can be inhibited in vitro by two sera from patients with autoimmune diseases. The first serum, designated anti-SS-B (or La), has antibodies directed against a 50,000 dalton polypeptide that is part of a larger ribonucleoprotein complex. The second serum, designated anti-SpNo, recognizes a target antigen polypeptide of greater than 100,000 daltons as well as the SS-B antigen. Both sera selectively remove required transcription factors from the transcription extract, and inhibition can be rescued by the addition of a HeLa S100 extract to the depleted transcription system. The HeLa S100 extract was sequentially fractionated by ion-exchange chromatography on DEAE-cellulose and phosphocellulose. The high salt eluate from the latter column was also able to rescue the anti-SS-B inhibition as was the immunoaffinity-purified SS-B ribonucleoprotein complex isolated from HeLa, Xenopus or rabbit thymus. Immunoblots of the active fractions indicated that all contained the SS-B immunoreactive polypeptide, but probes of replica filters for DNA-binding suggested that the transcription factor is not the SS-B antigen but a 64,000 dalton polypeptide component of the antigen ribonucleoprotein complex. SS-B is itself an RNA-binding protein and could be shown to bind nascent 5S RNA transcripts in vitro. Differential ammonium sulfate precipitation and DNA cellulose chromatography has confirmed that a group of 64-68 K dalton polypeptides are components of the SS-B ribonucleoprotein complex associated with transcription factor activity.

Autoantibodies↗
PubMed1984

Eukaryotic transcription complexes.

Accurate transcription of eukaryotic genes in vitro is preceeded by the assembly of the template DNA into stable DNA-protein complexes. Such complexes have been reported for genes transcribed by each of the three eukaryotic RNA polymerases. DNAase I protection or footprint assays have yielded information as to the sites of protein factor binding. These sites correlate with many of the sequences which have been implicated as promoter elements through analysis of deletion mutants. Stable transcription complexes are also formed in microinjected Xenopus oocytes and such complexes can be shown to exist in vivo in nuclei and chromatin. The propagation of active transcription complexes may prove to be a crucial element in cellular differentiation.

Animals↗
PubMed1983

Control of 5S RNA transcription in Xenopus somatic cell chromatin: activation with an oocyte extract.

A chromatin fraction enriched for Xenopus 5S RNA genes has been isolated by restriction endonuclease digestion and sucrose gradient velocity sedimentation. Soluble chromatin sedimenting at 70-80S contains approximately 50% of the oocyte-expressed 5S RNA genes and only 1.5-3% of total chromatin DNA; this represents a 15- to 30-fold purification of the 5S genes. Such chromatin isolated from somatic cells (blood and cultured kidney cells) retains the transcriptionally-inactive state of the oocyte-expressed 5S genes. Soluble chromatin from somatic cells prepared by micrococcal nuclease digestion also retains the inactive state of the oocyte-type 5S genes. It is likely that the level of chromatin structure responsible for inactivity of the oocyte genes in somatic cells is the nucleosome or short chains of nucleosomes and not supranucleosomal structures. The oocyte-type genes can be rendered transcriptionally active in somatic cell chromatin either by salt extraction of some chromosomal proteins or by treatment with the ion exchange resin Dowex A50W-X2. Alternatively, activation of these genes can be achieved by incubating somatic cell chromatin or nuclei with an extract prepared from Xenopus oocytes. This effect is not specific for 5S RNA genes as the transcription of other small RNAs (including pre-tRNA) is stimulated by the oocyte extract. The activating factor(s) is resistant to micrococcal nuclease, nondialyzable, heat labile and sensitive to trypsin; thus it is highly likely to be a protein or a group of proteins. Partial purification of the activating factor(s) has been achieved by ion exchange chromatography.

Animals↗
PubMed1983

5S rRNA gene transcription factor IIIA alters the helical configuration of DNA.

Relaxation of Xenopus 5S plasmid DNA (pX1o8) in the presence of transcription factor (TF) IIIA reduces the linking number of the DNA. Parallel experiments with plasmid pMB9 or cloned hepatitis B viral DNA indicate a degree of non-specific unwinding by TF; however, 60% of the effect observed for pX1o8 is due to specific interaction of TF IIIA with the 5S rRNA gene internal promoter sequence. The extent of unwinding (0.2-0.4 helical turn per TF IIIA binding site) is not consistent with the complete denaturation of the 50-base-pair TF binding site; however, it is consistent with a change in helix rotation, denaturation of 2-4 nucleotides per binding site, or DNA wrapping about a protein core. We show that proteins other than TF IIIA (bovine serum albumin and RNase) have no effect on the linking number of DNA when present during relaxation and that the unwinding activity associated with TF is heat labile. These results suggest that TF IIIA may facilitate transcription by altering the helical configuration of 5S DNA.

Animals↗
PubMed1980

Organization of 5S genes in chromatin of Xenopus laevis.

The chromatin organization of the genes coding for 5S RNA in Xenopus laevis has been investigated with restriction endonucleases and micrococcal nuclease. Digestion of nuclei from liver, kidney, blood and kidney cells maintained in culture with micrococcal nuclease reveals that these Xenopus cells and tissues have shorter nucleosome repeat lengths than the corresponding cells and tissues from other higher organisms. 5S genes are organized in nucleosomes with repeat lengths similar to those of the bulk chromatin in liver (178 bp) and cultured cells (165 bp); however, 5S gene chromatin in blood cells has a shorter nucleosome repeat (176 bp) than the bulk of the genome in these cells (184 bp). From an analysis of the 5S DNA fragments produced by extensive restriction endonuclease cleavage of chromatin in situ, no special arrangement of the nucleosomes with respect to the sequence of 5S DNA can be detected. The relative abundance of 5S gene multimers follows a Kuhn distribution, with about 57% of all HindIII sites cleaved. This suggests that HindIII sites can be cleaved both in the nucleosome core and linker regions.

Animals↗