Oocyte-type 5S Research Articles

Restricted specificity of Xenopus TFIIIA for transcription of somatic 5S rRNA genes.

Xenopus transcription factor IIIA (TFIIIA) is phosphorylated on serine-16 by CK2. Replacements with alanine or glutamic acid were made at this position in order to address the question of whether phosphorylation possibly influences the function of this factor. Neither substitution has an effect on the DNA or RNA binding activity of TFIIIA. The wild-type factor and the alanine variant activate transcription of somatic- and oocyte-type 5S rRNA genes in nuclear extract immunodepleted of endogenous TFIIIA. The glutamic acid variant (S16E) supports the transcription of somatic-type genes at levels comparable to those of wild-type TFIIIA; however, there is no transcription of the oocyte-type genes. This differential behavior of the phosphomimetic mutant protein is also observed in vivo when using early-stage embryos, where this mutant failed to activate transcription of the endogenous oocyte-type genes. Template exclusion assays establish that the S16E mutant binds to the oocyte-type 5S rRNA genes and recruits at least one other polymerase III transcription factor into an inactive complex. Phosphorylation of TFIIIA by CK2 may allow the factor to continue to act as a positive activator of the somatic-type genes and simultaneously as a repressor of the oocyte-type 5S rRNA genes, indicating that there is a mechanism that actively promotes repression of the oocyte-type genes at the end of oogenesis.

How do linker histones mediate differential gene expression?

In developing Xenopus laevis embryos the multiple-copy oocyte-type 5S RNA genes are progressively shut down. Results presented in three recent articles 1-3 together demonstrate that replacement of the cleavage stage linker histone B4 by somatic H1 leads to chromatosomes positioned directly over these genes and adjacent sequences so as to occlude the binding site for the critical transcription factor TFIIIA. In contrast, on the somatic-type 5S genes the somatic H1 positions chromatosomes about 65 bp further upstream, thereby leaving the TFIIIA binding site exposed and the genes active. The somatic linker histone thus functions as a specific gene repressor.

Both the 5S rRNA gene and the AT-rich flanks of xenopus laevis oocyte-type 5S rDNA repeat are required for histone H1-dependent repression of transcription of pol III-type genes in in vitro reconstituted chromatin.

Incorporation of somatic histone H1 into chromatin during embryogenesis of Xenopus laevis results in repression of transcription of the oocyte- but not the somatic-type 5S rRNA genes. We showed earlier that a similar effect of the H1 observed in chromatin reconstituted on circular plasmids in vitro depends on its binding to the AT-rich flanks of the oocyte-type 5S rRNA gene. H1 binding results in stabilization of nucleosomes within the oocyte 5S rDNA repeat comprising the 5S rRNA gene with flanks and in reorganization of chromatin on the entire plasmid DNA. Performing in vitro transcription on reconstituted minichromosome templates carrying the oocyte 5S rRNA gene placed in different arrangements and at different distances from the AT-rich flanks, we now establish that the above effects of H1 observed upon its binding to the AT-rich sequences are absolutely dependent on the presence of the 120 bp oocyte 5S rRNA gene in its native position within the flanks. We also find that with the intact oocyte 5S rDNA repeat, the binding of H1 results in repression of transcription of both pol III- and pol II-type genes and that the transcriptionally inactive chromatin state spreads over a distance of at least a few nucleosomes.

Inhibition of RNA polymerase III transcription by a ribosome-associated kinase activity.

Ribosomes prepared from somatic tissue of Xenopus laevis inhibit transcription by RNA polymerase III. This observation parallels an earlier report that a high speed fraction from activated egg extract, which is enrichedin ribosomes, inhibits RNA polymerase III activityand destabilizes putative transcription complexes assembled on oocyte 5S rRNA genes. Transcription of somatic- and oocyte-type 5S rRNA genes and a tRNA gene are all repressed in the present experiments. We find that 5S rRNA genes incubated in S150 extract prepared from immature oocytes exhibit an extensive DNase I protection pattern that is nearly identical to that of the ternary complex of TFIIIA and TFIIIC bound to a somatic 5S rRNA gene. The complexes formed in this extract are stable at concentrations of ribosomes that completely repress transcription, indicating that formation of the TFIII(A+C) complex is not the target of inhibition. Ribosomes taken through a high salt treatment no longer repress transcription of class III genes, establishing that the inhibition is due to an associated factor and not the particle itself. The inhibitory activity released from ribosomes is inactivated by treatment with proteinase K, but not micrococcal nuclease. Preincubation of ribosomes with a general protein kinase inhibitor, 6-dimethylaminopurine, eliminates repression of transcription. Western blot analysis demonstrates that p34(cdc2), which is known to mediate repression of transcription by RNA polymerase III, is present in these preparations of ribosomes and can be released from the particles upon extraction with high salt. These results establish that a kinase activity, possibly p34(cdc2), is the actual agent responsible for the observed inhibition of transcription by ribosomes.

Role of histone H1 as an architectural determinant of chromatin structure and as a specific repressor of transcription on Xenopus oocyte 5S rRNA genes.

We explore the role of histone H1 as a DNA sequence-dependent architectural determinant of chromatin structure and of transcriptional activity in chromatin. The Xenopus laevis oocyte- and somatic-type 5S rRNA genes are differentially transcribed in embryonic chromosomes in vivo depending on the incorporation of somatic histone H1 into chromatin. We establish that this effect can be reconstructed at the level of a single nucleosome. H1 selectively represses oocyte-type 5S rRNA genes by directing the stable positioning of a nucleosome such that transcription factors cannot bind to the gene. This effect does not occur on the somatic-type genes. Histone H1 binds to the 5' end of the nucleosome core on the somatic 5S rRNA gene, leaving key regulatory elements in the promoter accessible, while histone H1 binds to the 3' end of the nucleosome core on the oocyte 5S rRNA genes, specifically blocking access to a key promoter element (the C box). TFIIIA can bind to the somatic 5S rRNA gene assembled into a nucleosome in the presence of H1. Because H1 binds with equivalent affinities to nucleosomes containing either gene, we establish that it is the sequence-selective assembly of a specific repressive chromatin structure on the oocyte 5S rRNA genes that accounts for differential transcriptional repression. Thus, general components of chromatin can determine the assembly of specific regulatory nucleoprotein complexes.

Nucleolar targeting of 5S RNA in Xenopus laevis oocytes: somatic-type nucleotide substitutions enhance nucleolar localization.

In Xenopus laevis oocytes, 5S RNA is stored in the cytoplasm until vitellogenesis, at which time it is imported into the nucleus and targeted to nucleoli for ribosome assembly. This article shows that throughout oogenesis there is a pool of nuclear 5S RNA which is not nucleolar-associated. This distribution reflects that of oocyte-type 5S RNA, which is the major 5S RNA species in oocytes; only small amounts of somatic-type, which differs by six nucleotides, are synthesized. Indeed, 32P-labeled oocyte-type 5S RNA showed a degree of nucleolar localization similar to endogenous 5S RNA (33%) after microinjection. In contrast, 32P-labeled somatic-type 5S RNA showed significantly enhanced localization, whereby 70% of nuclear RNA was associated with nucleoli. A chimeric RNA molecule containing only one somatic-specific nucleotide substitution also showed enhanced localization, in addition to other somatic-specific phenotypes, including enhanced nuclear import and ribosome incorporation. The distribution of 35S-labeled ribosomal protein L5 was similar to that of oocyte-type 5S RNA, even when preassembled with somatic-type 5S RNA. The distribution of a series of 5S RNA mutants was also analyzed. These mutants showed various degrees of localization, suggesting that the efficiency of nucleolar targeting can be influenced by many discrete regions of the 5S RNA molecule.

The globular domain of histone H1 is sufficient to direct specific gene repression in early Xenopus embryos.

One molecule of a linker histone such as histone H1 is incorporated into every metazoan nucleosome [1]. Histone H1 has three distinct structural domains: the positively charged amino-terminal and carboxy-terminal tails are separated by a globular domain that is similar to the winged-helix motif found in sequence-specific DNA-binding proteins [2]. The globular domain interacts with DNA immediately contiguous to that wrapped around the core histones [3,4], whereas the tail domains are important for the compaction of nucleosomal arrays [5]. Experiments in vivo indicate that histone H1 does not function as a global transcriptional repressor, but instead has more specific regulatory roles [6-9]. In Xenopus, maternal stores of the B4 linker histone that are assembled into chromatin during the early cleavage divisions are replaced by somatic histone H1 during gastrulation [10]. This transition in chromatin composition causes the repression of genes encoding oocyte-type 5S rRNAs, and restricts the competence of ectodermal cells to differentiate into mesoderm [6,9-11]. Here, we demonstrate that the globular domain of histone H1 is sufficient for directing gene-specific transcriptional repression and for restricting the mesodermal competence of embryonic ectoderm. We discuss our results in the context of specific structural roles for this domain in the nucleosome.

The AT-rich flanks of the oocyte-type 5S RNA gene of Xenopus laevis act as a strong local signal for histone H1-mediated chromatin reorganization in vitro.

In vivo, histone H1 plays an active role in establishing the transcriptionally repressed chromatin state of the oocyte-type 5S RNA genes in the early stages of Xenopus development. By using fully defined in vitro system of chromatin assembly on plasmids with cloned oocyte- or somatic-type 5S gene repeats we found that the oocyte repeat which comprises a 120 bp oocyte-type 5S RNA gene placed within the few hundred bp long native AT-rich flanks, but not the somatic repeat (a similar 120 bp somatic-type 5S RNA gene placed within native GC-rich flanks) enables histone H1 to realign the nucleosomal core particles densely packed on plasmid DNA. The realignment results in creation of the repeat unit of approximately 240 bp and is achieved through complete removal of several core histone complexes from plasmid template with the oocyte-type repeat. This effect of H1 is independent on the plasmid sequences and seems to be solely due to the presence in the oocyte-repeat of the AT-rich flanks. The effects of H1 are completely suppressed by distamycin A, a drug that specifically recognizes and binds oligo(dA).oligo(dT) runs in DNA. The binding of H1 results in increased protection of DNA sites within the AT-rich oocyte-type 5S repeat. In an in vitro transcription assay performed with reconstituted chromatin templates containing plasmids with the oocyte- or somatic-type repeats only the transcription of the oocyte-type 5S RNA gene was repressed in the presence of physiological concentration of histone H1. These results support the view that the AT-rich flanks of the oocyte-type 5S RNA gene are involved in histone H1-mediated chromatin reorganization that results in the transcriptional repression observed in vivo.

Differential Binding of Oocyte-Type and Somatic-Type 5S rRNA to TFIIIA and Ribosomal Protein L5 in Xenopus Oocytes: Specialization for Storage versus Mobilization

We studied the pathway of 5S ribosomal RNA (rRNA) during oogenesis in Xenopus from its storage in the cytoplasm to incorporation into ribosomes in the nucleus. Ribonucleoprotein particle (RNP) assembly assays reveal striking differences in the Behavior of oocyte-type and somatic-type 5S rRNA after microinjection into stage II, III, or IV oocytes or into the cytoplasm of stage V-VI oocytes. Microinjected oocyte-type 5S rRNA predominantly interacts with the 5S rRNA gene-specific transcription factor IIIA (TFIIIA) to form storage 7S RNPs. In contrast, microinjected somatic-type 5S rRNA predominantly interacts with ribosomal protein L5 to form 5S RNPs, which are precursors to ribosome assembly. In addition, a greater amount of somatic-type 5S rRNA accumulates in the nucleus and is assembled into 60S ribosomal subunits. Thus, a slight difference in nucleotide sequence results in differential binding of 5S rRNA to TFIIIA and L5, specializing oocyte-type for storage in the oocyte cytoplasm and somatic-type for rapid mobilization and ribosome assembly. When oocyte-type and somatic-type 5S rRNA molecules were microinjected into the nucleus of stage V-VI oocytes in excess of other ribosomal components, the nucleocytoplasmic distribution of both types of RNA was similar, but the distinctive protein associations were maintained. In contrast, the behavior of oocyte-type and somatic-type 5S rRNA gradually synthesized in situ from microinjected cloned genes was similar, suggesting that nascent RNA is rapidly and directly recruited into ribosomes, thus bypassing an excursion into the cytoplasm prior to ribosome assembly.

The H1A histone variant is an in vivo repressor of oocyte-type 5S gene transcription in Xenopus laevis embryos.

Previous in vitro transcription studies have pointed to the importance of histone H1 for repression of oocyte-type 5S genes of Xenopus laevis. It has been previously reported that in development up to the early gastrula stage, Xenopus embryos contain a large pool of the maternal histone H1 variant H1M but are virtually devoid of histone H1A, H1B, and H1C proteins. At the early gastrula stage, there is an increase in H1A protein synthesis and H1A becomes the predominant H1 histone variant. Concomitant with the significant appearance of H1A protein in chromatin, oocyte 5S transcription is repressed. Here it is shown that there appears to be a direct link between H1A accumulation and inhibition of oocyte-type 5S RNA synthesis. Inhibition of H1A synthesis by a ribozyme targeted to H1A mRNA leads to the continued expression of oocyte 5S genes. H1A is proposed to inhibit major oocyte 5S gene transcription by sealing the nucleosome that is positioned over the major oocyte 5S coding sequences and by driving major oocyte 5S gene chromatin into a higher-order structure in which histone H1A molecules interact cooperatively.

Role of Maturation-Promoting Factor (p34cdc2-Cyclin B) in Differential Expression of the Xenopus Oocyte and Somatic-Type 5S RNA Genes

Transcription of 5S rRNA and tRNA genes by RNA polymerase III (pol III) in cytosolic extracts of unfertilized Xenopus eggs and in a reconstituted system derived from Xenopus oocytes is repressed by the action of one or more mitotic protein kinases. Repression is due to the phosphorylation of a component of the pol III transcription apparatus. We find that the maturation/mitosis-promoting factor kinase (MPF, p34cdc2-cyclin B) can directly mediate this repression in vitro. Affinity-purified MPF and immune complexes formed with antibodies to the protein subunits of MPF (p34cdc2 and cyclin B) retain both histone H1 kinase activity and the capacity to repress transcription in the reconstituted transcription system. Transcription complexes of oocyte-type 5S RNA genes and tRNA genes are quantitatively more sensitive to MPF repression than the corresponding transcription complexes of the somatic-type 5S RNA gene. The differential transcription of oocyte- and somatic-type genes observed during early Xenopus embryogenesis has been reproduced with the reconstituted transcription system and affinity-purified MPF. This differential transcription may be due to the instability of transcription complexes on the oocyte-type genes and the heightened sensitivity of soluble transcription factors to inactivation by mitotic phosphorylation. Our results suggest that MPF may play a role in vivo in the establishment of the embryonic pattern of pol III gene expression.

Role of maturation-promoting factor (p34cdc2-cyclin B) in differential expression of the Xenopus oocyte and somatic-type 5S RNA genes.

Transcription of 5S rRNA and tRNA genes by RNA polymerase III (pol III) in cytosolic extracts of unfertilized Xenopus eggs and in a reconstituted system derived from Xenopus oocytes is repressed by the action of one or more mitotic protein kinases. Repression is due to the phosphorylation of a component of the pol III transcription apparatus. We find that the maturation/mitosis-promoting factor kinase (MPF, p34cdc2-cyclin B) can directly mediate this repression in vitro. Affinity-purified MPF and immune complexes formed with antibodies to the protein subunits of MPF (p34cdc2 and cyclin B) retain both histone H1 kinase activity and the capacity to repress transcription in the reconstituted transcription system. Transcription complexes of oocyte-type 5S RNA genes and tRNA genes are quantitatively more sensitive to MPF repression than the corresponding transcription complexes of the somatic-type 5S RNA gene. The differential transcription of oocyte- and somatic-type genes observed during early Xenopus embryogenesis has been reproduced with the reconstituted transcription system and affinity-purified MPF. This differential transcription may be due to the instability of transcription complexes on the oocyte-type genes and the heightened sensitivity of soluble transcription factors to inactivation by mitotic phosphorylation. Our results suggest that MPF may play a role in vivo in the establishment of the embryonic pattern of pol III gene expression.

Structural Requirements of 5S rRNA for Nuclear Transport, 7S Ribonucleoprotein Particle Assembly, and 60S Ribosomal Subunit Assembly inXenopusOocytes

Structural requirements of 5S rRNA for nuclear transport and RNA-protein interactions have been studied by analyzing the behavior of oocyte-type 5S rRNA and of 31 different in vitro-generated mutant transcripts after microinjection into the cytoplasm of Xenopus oocytes. Experiments reveal that the sequence and secondary and/or tertiary structure requirements of 5S rRNA for nuclear transport, storage in the cytoplasm as 7S ribonucleoprotein particles, and assembly into 60S ribosomal subunits are complex and nonidentical. Elements of loops A, C, and E, helices II and V, and bulged and hinge nucleotides in the central domain of 5S rRNA carry the essential information for these functional activities. Assembly of microinjected 5S rRNA into 60S ribosomal subunits was shown to occur in the nucleus; thus, the first requirement for subunit assembly is nuclear targeting. The inhibitory effects of ATP depletion, wheat germ agglutinin, and chilling on the nuclear import of 5S rRNA indicate that it crosses the nuclear envelope through the nuclear pore complex by a pathway similar to that used by karyophilic proteins.

Structural requirements of 5S rRNA for nuclear transport, 7S ribonucleoprotein particle assembly, and 60S ribosomal subunit assembly in Xenopus oocytes.

Structural requirements of 5S rRNA for nuclear transport and RNA-protein interactions have been studied by analyzing the behavior of oocyte-type 5S rRNA and of 31 different in vitro-generated mutant transcripts after microinjection into the cytoplasm of Xenopus oocytes. Experiments reveal that the sequence and secondary and/or tertiary structure requirements of 5S rRNA for nuclear transport, storage in the cytoplasm as 7S ribonucleoprotein particles, and assembly into 60S ribosomal subunits are complex and nonidentical. Elements of loops A, C, and E, helices II and V, and bulged and hinge nucleotides in the central domain of 5S rRNA carry the essential information for these functional activities. Assembly of microinjected 5S rRNA into 60S ribosomal subunits was shown to occur in the nucleus; thus, the first requirement for subunit assembly is nuclear targeting. The inhibitory effects of ATP depletion, wheat germ agglutinin, and chilling on the nuclear import of 5S rRNA indicate that it crosses the nuclear envelope through the nuclear pore complex by a pathway similar to that used by karyophilic proteins.

Role of TFIIIA zinc fingers in vivo: analysis of single-finger function in developing Xenopus embryos.

The Xenopus 5S RNA gene-specific transcription factor IIIA (TFIIIA) has nine consecutive Cys2His2 zinc finger motifs. Studies were conducted in vivo to determine the contribution of each of the nine zinc fingers to the activity of TFIIIA in living cells. Nine separate TFIIIA mutants were expressed in Xenopus embryos following microinjection of their respective in vitro-derived mRNAs. Each mutant contained a single histidine-to-asparagine substitution in the third zinc ligand position of an individual zinc finger. These mutations result in structural disruption of the mutated finger with little or no effect on the other fingers. The activity of mutant proteins in vivo was assessed by measuring transcriptional activation of the endogenous 5S RNA genes. Mutants containing a substitution in zinc finger 1, 2, or 3 activate 5S RNA genes at a level which is reduced relative to that in embryos injected with the message for wild-type TFIIIA. Proteins with a histidine-to-asparagine substitution in zinc finger 5 or 7 activate 5S RNA genes at a level that is roughly equivalent to that of the wild-type protein. Zinc fingers 8 and 9 appear to be critical for the normal function of TFIIIA, since mutations in these fingers result in little or no activation of the endogenous 5S RNA genes. Surprisingly, proteins with a mutation in zinc finger 4 or 6 stimulate 5S RNA transcription at a level that is significantly higher than that mediated by similar concentrations of wild-type TFIIIA. Differences in the amount of newly synthesized 5S RNA in embryos containing the various mutant forms of TFIIIA result from differences in the relative number and/or activity of transcription complexes assembled on the endogenous 5S RNA genes and, in the case of the finger 4 and finger 6 mutants, result from increased transcriptional activation of the normally inactive oocyte-type 5S RNA genes. The remarkably high activity of the finger 6 mutant can be reproduced in vitro when transcription is carried out in the presence of 5S RNA. Disruption of zinc finger 6 results in a form of TFIIIA that exhibits reduced susceptibility to feedback inhibition by 5S RNA and therefore increases the availability of the transcription factor for transcription complex formation.

Role of TFIIIA zinc fingers in vivo: analysis of single-finger function in developing Xenopus embryos.

The Xenopus 5S RNA gene-specific transcription factor IIIA (TFIIIA) has nine consecutive Cys2His2 zinc finger motifs. Studies were conducted in vivo to determine the contribution of each of the nine zinc fingers to the activity of TFIIIA in living cells. Nine separate TFIIIA mutants were expressed in Xenopus embryos following microinjection of their respective in vitro-derived mRNAs. Each mutant contained a single histidine-to-asparagine substitution in the third zinc ligand position of an individual zinc finger. These mutations result in structural disruption of the mutated finger with little or no effect on the other fingers. The activity of mutant proteins in vivo was assessed by measuring transcriptional activation of the endogenous 5S RNA genes. Mutants containing a substitution in zinc finger 1, 2, or 3 activate 5S RNA genes at a level which is reduced relative to that in embryos injected with the message for wild-type TFIIIA. Proteins with a histidine-to-asparagine substitution in zinc finger 5 or 7 activate 5S RNA genes at a level that is roughly equivalent to that of the wild-type protein. Zinc fingers 8 and 9 appear to be critical for the normal function of TFIIIA, since mutations in these fingers result in little or no activation of the endogenous 5S RNA genes. Surprisingly, proteins with a mutation in zinc finger 4 or 6 stimulate 5S RNA transcription at a level that is significantly higher than that mediated by similar concentrations of wild-type TFIIIA. Differences in the amount of newly synthesized 5S RNA in embryos containing the various mutant forms of TFIIIA result from differences in the relative number and/or activity of transcription complexes assembled on the endogenous 5S RNA genes and, in the case of the finger 4 and finger 6 mutants, result from increased transcriptional activation of the normally inactive oocyte-type 5S RNA genes. The remarkably high activity of the finger 6 mutant can be reproduced in vitro when transcription is carried out in the presence of 5S RNA. Disruption of zinc finger 6 results in a form of TFIIIA that exhibits reduced susceptibility to feedback inhibition by 5S RNA and therefore increases the availability of the transcription factor for transcription complex formation.

Coordinate inactivation of class III genes during the gastrula-neurula transition in Xenopus

We have identified a period during early Xenopus development when several different genes transcribed by RNA polymerase III (class III genes) are coordinately inactivated. During the late gastrula stage a major reduction in the number of active transcription complexes gives rise to a pattern of class III gene activity typical of adult somatic cells. This event is referred to as the Gastrula-Neurula Transition and involves the inactivation of genes encoding oocyte-type tRNAs and 5S RNA, along with several heterogeneous RNAs expressed during the blastula and gastrula stages of embryogenesis.

TFIIIA induced DNA bending: effect of low ionic strength electrophoresis buffer conditions

We have used a circular permutation gel shift assay to show that the 5S gene transcription factor, TFIIIA, induces a bend at the internal promoter of the Xenopus oocyte-type 5S gene. The degree of bending is comparable to what we have previously observed for TFIIIA induced bending of the Xenopus somatic-type gene [Schroth, G.P. et al. (1989) Nature 340, 487-488]. In addition, we show that TFIIIA induced DNA bending is dramatically affected by the ionic conditions used during gel electrophoresis. By modifying the conditions of the electrophoresis, we can detect two distinct conformations for the TFIIIA/DNA complex. In very low ionic strength buffers, the degree of DNA bending in the complex is estimated to be about 25 to 30 degrees, whereas in higher ionic strength buffers it is about 60 to 65 degrees. These data explain the apparent discrepancy between our results and the results of another study in which it was claimed that TFIIIA did not 'substantially' bend DNA [Zweib, C. and Brown, R.S. (1990) Nucleic Acid Res. 18, 583-587]. These results also demonstrate that the TFIIIA/DNA complex has a large degree of conformational flexibility. Both DNA bending and conformational flexibility are structural features which may provide a key insight into the function of TFIIIA as a positive transcription factor.

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.

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.