Somatic 5S RNA Gene Research Articles

Determination of the chromatin domain structure in arrayed repeat regions: Organization of the somatic 5S RNA domain during embryogenesis inXenopus laevis

The size of the DNA loop containing the Xenopus laevis somatic 5S RNA gene cluster has been estimated using a simple, precise and sensitive method that we have developed for use on any tandemly arrayed DNA repeat region, and was found to increase during development We have found that after the mid-blastula transition, when transcription is activated in the embryo, a subset of somatic 5S RNA genes becomes specifically associated with the nuclear matrix. This association correlates with the transcriptional activity of the 5S genes.

The third zinc finger of TFIIIA stabilizes a hairpin structure of the non-coding strand in the internal control region of 5S RNA gene

The structures of non-coding and coding strands in box C of the internal control region (ICR) of Xenopus laevis somatic 5S RNA gene have been examined by circular dichroism (CD) and Raman spectroscopy in the absence and presence of the third zinc finger of transcription factor IIIA (TFIIIA), which binds to the ICR. The non-coding strand exhibits CD signals assignable to a hairpin and an unfolded structure. The presence of the hairpin structure is supported by Raman spectra, gel electrophoresis, and nucleotide deletion experiments. Binding of the zinc finger to the non-coding strand increases the CD signal of hairpin structure, indicating stabilization of the hairpin structure by the zinc finger. In contrast, the corresponding coding strand remains unfolded even in the presence of the zinc finger. The TFIIIA–ICR complex is not only required for initiation of transcription but also lasts during many rounds of transcription of the 5S RNA gene including the ICR (Bogenhagen et al., Cell 28 (1982) 413). TFIIIA may play a role in promoting the transcription by maintaining the unwound non-coding strand in the hairpin structure and leaving the coding strand available for transcription by RNA polymerase.

Enhancer of RNA polymerase III gene transcription.

A protein responsible for enhanced transcription by RNA polymerase III was identified in extracts from Xenopus oocytes. This protein, called EP3, interacts with a specific DNA sequence adjacent to the 3'-end of a Xenopus somatic 5S RNA gene and forms a distinct band shift complex with a unique DNase I footprint. Enhanced transcription was observed from both 5S RNA and tRNA reporter genes when EP3 binding sites were inserted at different locations and orientations. Removal of the EP3 protein from an oocyte extract abolished this enhanced transcription. In addition, EP3 was shown to stimulate transcription by increasing the rate of transcription complex assembly. EP3 directly discriminates between the somatic and oocyte 5S RNA gene families and may play a significant role in their differential expression during early Xenopus development.

Expanded CTG triplet blocks from the myotonic dystrophy gene create the strongest known natural nucleosome positioning elements

Expanded blocks of repeating nucleotide triplets have been found in or near genes associated with several human diseases. In the case of myotonic dystrophy, a block of repeating CTG trinucleotides is located downstream of the gene, and expansions of this block to repeats of n = 100 or more are frequently found in afflicted individuals. Using electron microscopy, we recently demonstrated that these blocks form unusually stable nucleosomes. Here, competitive nucleosome reconstitution was employed to measure the energetics of nucleosome formation over CTG repeat blocks of n = 75 and n = 130. These values were compared to the Xenopus borealis somatic 5S RNA gene, previously one of the strongest known natural nucleosome positioning elements. It is shown that DNA fragments containing 75 and 130 CTG repeats are 6 and 9 times stronger in nucleosome formation, respectively, than the 5S RNA gene. These findings suggest that expanded CTG blocks may profoundly alter local chromatin structure.

Contacts of the globular domain of histone H5 and core histones with DNA in a "chromatosome".

The globular domain of histone H5 is found to asymmetrically associate with a nucleosome core including the Xenopus borealis somatic 5S RNA gene. Histones H2A and H2B are required for association of histone H5. Strong crosslinking of the globular domain of histone H5 to the 5S DNA in the nucleosome occurs at a single site to one side of the dyad axis. This site is also in contact with the core histones, and the interactions of the core histones with 5S DNA change as a result of association of the globular domain of histone H5. We discuss evidence for an allosteric change in core histone-5S DNA interactions following the association of the linker histone in the nucleosome.

Preferential and asymmetric interaction of linker histones with 5S DNA in the nucleosome.

We establish that linker histones H1 and H5 bind preferentially to a Xenopus borealis somatic 5S RNA gene associated with an octamer of core histones rather than to naked 5S DNA. This preferential binding requires free linker DNA to either side of the nucleosome core. Incorporation of a single linker histone molecule into the nucleosome protects an additional 20 bp of linker DNA from micrococcal nuclease digestion. This additional DNA is asymmetrically distributed with respect to the nucleosome core. Incorporation of linker histones causes no change to the cleavage of DNA in the nucleosome by hydroxyl radical or DNase I.

Replication Timing and Xenopus 5S RNA Gene Transcription in Vitro

The influence of DNA replication in a chromospinal environment on the transcription of Xenopus laevis 5S RNA genes has been examined using low-speed extracts of Xenopus eggs and Xenopus sperm nuclei. The major oocyte- and somatic-type 5S RNA multigene families of X. laevis are differentially expressed in these extracts dependent on limiting transcription factors and replicate in different compartments of S-phase. Under these particular experimental conditions the timing of replication of the somatic 5S RNA genes appears to be independent of their transcriptional activity. Moreover, differential transcription of the oocyte- and somatic-type 5S RNA genes is not influenced by the replication process.

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.

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.

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.

Binding of TFIIIA to derivatives of 5S RNA containing sequence substitutions or deletions defines a minimal TFIIIA binding site.

The repetitive zinc finger domain of transcription factor IIIA binds 5S DNA and 5S RNA with similar affinity. Site directed mutagenesis of the Xenopus borealis somatic 5S RNA gene has been used to produce a series of derivatives of 5S RNA containing local sequence substitutions or sequence deletions. Gel mobility shift analyses of the binding of TFIIIA to these altered 5S RNAs revealed that all three of the helical stems of the 5S RNA secondary structure are required for binding. TFIIIA was observed to bind with normal affinity to RNAs lacking 12 nucleotides at either the loop c or loop e/helix V regions of 5S RNA, as well as to a double mutant containing both deletions. The secondary structure of the resulting 96-nucleotide RNA, studied using structure-specific ribonucleases, was found to resemble the central portion of 5S RNA.

Acetylaminofluorene and aminofluorene adducts inhibit in vitro transcription of a Xenopus 5S RNA gene only when located on the coding strand.

Unique N-acetyl-2-aminofluorene (AAF) or 2-aminofluorene (AF) adducts were introduced into the Xenopus borealis somatic 5S RNA gene between the intragenic control region and the transcription termination site. The effects of these bulky adducts on transcription were studied in a cell-free extract derived from Xenopus laevis oocytes. AAF and AF adducts inhibit transcription only when they are on the template strand, whereas transcription passes through these adducts when they are placed on the nontemplate strand. In the presence of the AAF or AF adduct on the template strand, transcription usually terminates one nucleotide before the altered guanine residue. Premature termination at these bulky adducts does not block reinitiation of transcription, since several transcripts are produced per gene per hour on these damaged templates.

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.

A 3' exonuclease activity degrades the pseudogene 5S RNA transcript and processes the major oocyte 5S RNA transcript in Xenopus oocytes.

Transcription of the major oocyte 5S RNA gene (o) and pseudogene (psi) of Xenopus laevis yields different RNAs with three different homologous systems: oocyte microinjection, whole oocyte extract, and fractionated TFIIIA + TFIIIB + TFIIIC components. Those peculiar results are caused by a 3' RNA exonuclease activity, which is inhibited in the oocyte extract, that rapidly degrades the pseudogene 5S RNA but does not degrade as readily the chimeric RNA transcripts generated by HindIII-truncated 5S RNA pseudogenes. The same, or a similar, RNase activity processes the 130- and the 142-base-long transcripts of the major oocyte 5S RNA gene into mature 120-base-long 5S RNA. We performed site-specific mutagenesis on the somatic 5S RNA gene and changed specific nucleotides on the somatic 5S RNA. These studies indicated that the structure that confers stability to the 5S RNA in vivo and in vitro is the 9-bp helix formed in 5S RNA, but not in psi 5S RNA, by the complementary 5' and 3' ends of the molecule.

Effect of sequence differences between somatic and oocyte 5S RNA genes on transcriptional efficiency in an oocyte S150 extract.

The differential expression of the Xenopus laevis somatic and oocyte 5S RNA genes is partially, but not solely, due to several base differences near the 5' boundary of the internal control region. A hybrid oocyte 5S gene with somatic-type base changes at +47, +53, +55, and +56 had intermediate transcriptional activity in oocyte S150 extracts. These base substitutions also resulted in increased affinity for a factor(s), other than TFIIIA, which forms a stable complex with the 5S gene.

Developmental Regulation of Two 5 S Ribosomal RNA Genes

The developmental regulation of two kinds of Xenopus 5S RNA genes (oocyte and somatic types) can be explained by differences in the stability of protein-protein and protein-DNA interactions in a transcription complex that directs transcription initiation by RNA polymerase III. Dissociation of transcription factors from oocyte 5S RNA genes during development allows them to be repressed by chromatin assembly. In the same cells, somatic 5S RNA genes remain active because their transcription complexes are stable.

Transcription complexes that program Xenopus 5S RNA genes are stable in vivo.

The long-term stability of transcription complexes on 5S RNA genes has been demonstrated in vivo. Complexes on oocyte and somatic-type 5S RNA genes injected into Xenopus laevis oocyte nuclei are stable for at least 4 days. Tissue culture cells and mature erythrocytes have equivalent numbers of somatic 5S RNA genes programmed into transcription complexes, yet the former cell type has a greater than 50-fold higher cellular content of transcription factor IIIA (TFIIIA). Functional transcription complexes on somatic 5S RNA genes in nucleated erythrocytes of Xenopus are stable for weeks, perhaps months, even though a mature erythrocyte has less than two molecules of TFIIIA for each somatic 5S RNA gene. These findings strengthen our proposal that stable transcription complexes are a means of maintaining the differentiated state.

Transcription fraction TFIIIC can regulate differential Xenopus 5S RNA gene transcription in vitro.

An extract of whole oocytes (oocyte S150) differentially transcribes Xenopus oocyte and somatic 5S RNA genes. In the oocyte S150, transcription complexes with different stabilities are assembled onto oocyte and somatic 5S DNA. The stability of the transcription complex is dependent on activities present in a fraction containing transcription factor TFIIIC. This fraction stabilizes the binding of the positive transcription factor TFIIIA to a somatic 5S RNA gene much more efficiently than it does to an oocyte gene. The oocyte S150 transcription extract is deficient in TFIIIC such that supplementation with a fraction enriched in this transcription factor selectively stimulates oocyte 5S DNA transcription. Previously it has been shown that an egg extract deficient in TFIIIA selectively transcribes somatic 5S RNA genes. Thus under conditions where there is differential stability of transcription complexes, limitation of either TFIIIA or TFIIIC may exaggerate the differential expression of two genes.

TFIIIA binds with equal affinity to somatic and major oocyte 5S RNA genes.

Current models for the differential control of expression of Xenopus somatic and oocyte 5S RNA genes suggest that an impaired ability to bind TFIIIA contributes to the inactivation of oocyte 5S RNA genes in somatic cells. The somatic 5S RNA gene is transcribed more efficiently than the major oocyte 5S RNA gene in S-150 extracts of mature oocytes. However, this differential transcription efficiency is not determined simply by the relative affinity for binding of a positive transcription factor, TFIIIA. We have compared the abilities of somatic, major oocyte, and minor oocyte 5S RNA genes to bind TFIIIA using both a standard footprint competition assay and an indirect DNase protection assay. This indirect DNase protection assay permits the direct comparison of TFIIIA binding to two templates in one reaction. Both assay methods indicate that the major oocyte 5S RNA gene and the somatic 5S RNA gene bind TFIIIA with equal affinity. As a further control, we have confirmed earlier work indicating that the minor oocyte gene binds TFIIIA with a reduced affinity. Binding of TFIIIA to these three 5S RNA genes results in a different pattern of protection of each gene. We suggest that slight differences in the contacts between TFIIIA and the 5' border of the control region influence the ability of additional transcription factors to bind to the TFIIIA:5S DNA complex.

Differential 5S RNA gene expression in vitro

In extracts derived from activated eggs of Xenopus, oocyte 5S RNA gene transcription complexes are destabilized under conditions where somatic 5S RNA gene transcription complexes are stable. Trans-acting factors in destabilized complexes are in equilibrium with free factors in the extract, and thus transcription becomes dependent upon the concentration of the limiting factor in the extract. By reducing TFIIIA, a limiting factor in the egg extract, a cloned somatic 5S RNA gene is transcribed as much as 400-fold more efficiently than a cloned oocyte 5S RNA gene.