Somatic 5S RNA Gene Research Articles

Transition mutations within the Xenopus borealis somatic 5S RNA gene can have independent effects on transcription and TFIIIA binding.

Base-pair changes were introduced into the Xenopus borealis somatic 5S RNA gene by treatment with sodium bisulfite. Mutants were screened by sequence determination. The collection of mutants permitted a detailed investigation of the fine structure of the intragenic control region that binds the transcription factor TFIIIA. Selected mutants were recloned in tandem with a somatic 5S RNA maxigene to permit sensitive measurement of their relative transcription activities. The transcription efficiencies of a number of mutations at the borders of the control region were correlated with TFIIIA binding by using DNase I protection (footprinting) assays. Mutations affecting transcription and TFIIIA binding extended from gene residues 46 to 91. The results reinforce a model in which the distal half of the protected region constitutes a tight binding domain for TFIIIA. A number of transitions in the 5' domain led to significant increases or decreases in transcription efficiency, but resulted in barely detectable changes in TFIIIA binding. Two mutants with C----T transitions at gene residues 52 and 53 were transcribed with increased efficiencies (up phenotype). These results suggest that TFIIIA must make appropriate contacts within the 5' domain of the control region to permit subsequent binding of other factors in stable transcription complexes.

Transition mutations within the Xenopus borealis somatic 5S RNA gene can have independent effects on transcription and TFIIIA binding.

Base-pair changes were introduced into the Xenopus borealis somatic 5S RNA gene by treatment with sodium bisulfite. Mutants were screened by sequence determination. The collection of mutants permitted a detailed investigation of the fine structure of the intragenic control region that binds the transcription factor TFIIIA. Selected mutants were recloned in tandem with a somatic 5S RNA maxigene to permit sensitive measurement of their relative transcription activities. The transcription efficiencies of a number of mutations at the borders of the control region were correlated with TFIIIA binding by using DNase I protection (footprinting) assays. Mutations affecting transcription and TFIIIA binding extended from gene residues 46 to 91. The results reinforce a model in which the distal half of the protected region constitutes a tight binding domain for TFIIIA. A number of transitions in the 5' domain led to significant increases or decreases in transcription efficiency, but resulted in barely detectable changes in TFIIIA binding. Two mutants with C----T transitions at gene residues 52 and 53 were transcribed with increased efficiencies (up phenotype). These results suggest that TFIIIA must make appropriate contacts within the 5' domain of the control region to permit subsequent binding of other factors in stable transcription complexes.

The 5S gene internal control region is composed of three distinct sequence elements, organized as two functional domains with variable spacing

Systematic oligonucleotide-directed mutagenesis within the internal control region of the Xenopus laevis somatic 5S RNA gene identifies three distinct sequence elements that regulate transcription activity: box A, containing the common, conserved class III promoter domain, and two 5S-gene-specific segments, termed intermediate element and box C. Analysis of the individual steps in the formation of the stable initiation complex reveals that the two 5S-gene-specific elements are the main determinants for the stable binding of TFIIIA. In contrast, TFIIIC binding appears to be dependent on interactions with TFIIIA and on direct DNA interactions in box A as well as probably in box C. Alterations of the spacing between the two major promoter domains of from −3 to +10 nucleotides are tolerated, although they reduce transcription activity and were found to prevent the formation of a stable initiation complex.

The developmental expression of the gene for TFIIIA in Xenopus laevis.

The sequence of the trans-acting positive transcription factor TFIIIA has been deduced by sequencing cDNA and genomic DNA clones. Using DNA from a homozygous diploid animal we show that there is one gene for TFIIIA per haploid genome of X. laevis. Protein sequencing of proteolytic fragments of TFIIIA orients the protein in its interaction with the internal control region (ICR) of the 5S RNA gene. The protein lies along the DNA with its carboxyl terminus at the 5' end of the ICR and its amino terminus at the 3' end. The developmental pattern of TFIIIA and its mRNA during oogenesis and embryogenesis are consistent with the idea that the abundance of TFIIIA plays an essential role in the developmental change in 5S RNA gene expression. The change to almost exclusive somatic 5S RNA gene expression by gastrulation occurs using either the TFIIIA that was synthesized by oocyte and/or TFIIIA synthesized from maternal mRNA.

A positive transcription factor controls the differential expression of two 5S RNA genes

Somatic 5S RNA genes are transcribed preferentially over oocyte 5S RNA genes by 25- to greater than 200-fold when mixtures of cloned genes are injected into cleaving Xenopus embryos. This preference is an order of magnitude greater than that observed in cell-free extracts. Mutations that decrease the binding of the 5S RNA gene-specific transcription factor TFIIIA to the 5S RNA genes' internal control regions are exaggerated when assayed by embryo injection compared to in vitro transcription. Injection of TFIIIA into cleaving embryos greatly increases endogenous oocyte 5S RNA synthesis at midblastula even when DNA replication is inhibited. Much, but not all, of the preference for somatic over oocyte 5S RNA gene transcription in somatic cells can thus be attributed to the concentration of TFIIIA and to differences in binding constants of TFIIIA to the internal control regions of the two types of genes.

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.

The transcriptional regulation of Xenopus 5S RNA genes in chromatin: The roles of active stable transcription complexes and histone H1

The properties of active and repressed 5S RNA genes in somatic cell chromatin from Xenopus laevis cultured cells were studied by transcription in vitro. The somatic 5S RNA genes, which are active in vivo, are packaged in chromatin as active stable transcription complexes lacking only RNA polymerase III for transcription activity. The oocyte 5S RNA genes, which are inactive in somatic tissues, do not have transcription factors bound to them in purified chromatin and are prevented from binding these factors by a structure dependent on histone H1. In chromatin active stable transcription complexes protect 5S RNA genes from histone H1-mediated repression and H1 binding prevents the formation of stable active transcription complexes.

A simple procedure for parallel sequence analysis of both strands of 5′-labeled DNA

Ligation of a 5′-labeled DNA restriction fragment results in a circular DNA molecule carrying the two 32Ps at the reformed restriction site. Double digestions of the circular DNA with the original enzyme and a second restriction enzyme cleavage near the labeled site allows direct chemical sequencing of one 5′-labeled DNA strand. Similar double digestions, using an isoschizomer that cleaves differently at the 32P-labeled site, allows direct sequencing of the now 3′-labeled complementary DNA strand. It is possible to directly sequence both strands of cloned DNA inserts by using the above protocol and a multiple cloning site vector that provides the necessary restriction sites. The simultaneous and parallel visualization of both DNA strands eliminates sequence ambiguities. In addition, the labeled circular molecules are particularly useful for single-hit DNA cleavage studies and DNA footprint analysis. As an example, we show here an analysis of the micrococcal nuclease-induced breaks on the two strands of the somatic 5S RNA gene of Xenopus borealis, which suggests that the enzyme may recognize and cleave small AT-containing palindromes along the DNA helix.

Cooperative model for the binding of Xenopus transcription factor A to the 5S RNA gene.

7S particles containing 5S RNA and the transcription regulatory protein factor A have been purified to near homogeneity from Xenopus laevis oocytes. The binding of the transcription factor to the Xenopus borealis somatic 5S RNA gene has been monitored by quantitating the DNase I protection patterns of the protein-DNA interaction. Under stoichiometric binding conditions--i.e., when all added DNA binds to the factor--two protein molecules are required to saturate the 5S RNA gene. Under equilibrium binding conditions, titration of the 5S RNA gene with factor A results in a sigmoidal binding isotherm suggesting a cooperative interaction; half-saturation of binding is observed at a free factor A concentration of 1 nM. Cooperative binding between factor A and the 5S RNA gene may contribute to the stability of the transcription apparatus and its maintenance during cell division.

Common and interchangeable elements in the promoters of genes transcribed by RNA polymerase III

We have shown that the 34 bp internal control region of the somatic 5S RNA gene from Xenopus borealis can be split into two separable components. A maxigene carrying an insertion between nucleotide 71 and nucleotide 74 of the coding region is actively transcribed in the nucleus of X. laevis oocytes, giving rise to a maxitranscript with initiation and termination points identical with those of the wild-type transcript. The first 11 bases of the 5S RNA gene promoter are shown to be structurally and functionally homologous with the first component (box A) of the promoter for tRNA genes. This was shown by constructing hybrid 5S RNA-tRNA Pro and tRNA Pro-5S RNA genes that were efficiently transcribed in the X. laevis oocytes. Initiation of transcription appears to be a complex phenomenon in which both components of the internal promoter play a role.

Contact points between a positive transcription factor and the Xenopus 5S RNA gene.

We have investigated the contact points of a positive transcription factor with the internal control region of the 5S ribosomal RNA genes of Xenopus. The methylation of any one of eight G residues clustered at the 3' end of the internal control region on the noncoding strand of the DNA or the ethylation of their surrounding phosphates interferes with the binding of this protein. The importance of the 5' half of the control region is shown by a binding affinity of the protein to oocyte 5S RNA genes that is one fourth that to somatic 5S RNA genes. This can be attributed to two base changes at residues 53 and 55 in the 5' part of the control region. The different affinities of the transcription factor for the oocyte and somatic 5S RNA genes may contribute to their differential expression in somatic cells. The almost exclusive location of strong contact points on the noncoding strand of the gene suggests how transcription events could proceed repeatedly along a gene that is assembled into a stable transcription complex. It is proposed that the complex is transiently shifted to the noncoding strand as each round of RNA synthesis occurs from the coding strand.

Stable transcription complexes of Xenopus 5S RNA genes: A means to maintain the differentiated state

Cloned 5S RNA genes added to a Xenopus oocyte nuclear extract assemble into stable active transcription complexes that persist for many rounds of 5S RNA synthesis. This stability of the complex has been demonstrated by its resistance to dilution and to competitor DNA. A stable complex is formed within minutes and lasts for at least 40 rounds of transcription per template over several hours. Stable, transcriptionally inactive complexes can be formed by incubation of cloned 5S RNA genes in an oocyte nuclear extract depleted of a 5S-specific transcription factor and supplemented with histones. The stable, transcriptionally active and inactive states of 5S RNA gene complexes that can be formed in vitro are analogous to the states of the somatic and oocyte 5S RNA genes as they exist in somatic cell chromatin. Oocyte 5S RNA genes remain repressed in chromatin isolated from somatic cells, but can be activated by washing chromatin with high salt. Maintenance of the differentiated state of a cell requires that selected genes remain stably active while others are stably repressed for long periods of time. We proposed that stable transcription complexes may play an important role in the maintenance of the differentiated state in eucaryotic cells.

Related 5S RNA transcription factors in Xenopus oocytes and somatic cells.

Xenopus oocytes contain an abundant protein that binds specifically to the center of 5S RNA genes and directs their transcription by RNA polymerase III. This protein also binds to 5S RNA. We show here that transcription of cloned 5S RNA genes in extracts derived from Xenopus tissue culture cells is dependent on the intragenic control region and is inhibited by 5S RNA and by antibodies raised against the previously characterized oocyte transcription factor. Somatic cells contain a protein that is similar to the oocyte factor in charge, affinity for heparin-agarose, and antigenicity but has an apparent molecular mass about 2000 daltons greater than that of the oocyte protein. Our experiments strongly suggest that this larger protein is the transcription factor for 5S RNA genes in somatic cells. The 5S RNA may regulate its own synthesis in somatic cells by binding to this protein, which is present at a low concentration. The presence of two different proteins responsible for 5S RNA synthesis in oocytes and in somatic cells cannot by itself explain the developmental control of oocyte and somatic 5S RNA genes, because somatic cell extracts transcribe both types of gene.

A control region in the center of the 5S RNA gene directs specific initiation of transcription: II. The 3′ border of the region

A control region that directs accurate initiation of 5S RNA synthesis is located within the middle third of the X. borealis somatic 5S RNA gene. The 5′ boundary of this region is described in the accompanying paper ( Sakonju, Bogenhagen and Brown, 1980) as occurring between residues 50 and 55 of the 120 nucleotide 5S RNA gene. In this paper we delimit the 3′ boundary of this control region between gene residues 80 and 83. The 3′ boundary has been defined by in vitro transcription of a series of deletions of cloned 5S DNA whose endpoints approach and enter the gene from the 3′ side. Since these deletions lack the normal termination site of 5S RNA synthesis, we have developed an assay for accurate initiation of transcription in the absence of correct termination. In vitro transcription in the presence of cordycepin triphosphate (3′ dATP) causes synthesis of a reproducible array of shortened RNA molecules displayed by polyacrylamide gel electrophoresis. This transcription assay shows that 3′ deletions leaving the first 83 or more 5′ gene residues support accurate initiation of transcription. Larger deletions leaving 80 or fewer gene residues do not support 5S RNA-specific transcription initiation. The gene region from nucleotides 41–87 which contains this control region has been cloned without any additional 5S DNA sequences. This internal gene fragment contains sufficient information to direct specific transcription initiation at a site in the upstream plasmid sequence.

Sequences of 5S ribosomal RNA from Xenopus mulleri and the evolution of 5S gene-coding sequences

Sequences for 5S RNA from somatic cells and oocytes of Xenopus mulleri are presented. Comparison with sequences previously given for Xenopus laevis indicates that the somatic 5S RNA genes of each species are more closely related to each other than either is to its own set of oocyte genes, suggesting that somatic and oocyte genes within each species are evolving independently. However, detailed analysis of sequence variants in each species suggests that there is a mechanism which allows occasional genetic exchanges between somatic and oocyte-specific genes. Possible genetic mechanisms which allow such an exchange are discussed.