An extract from whole oocytes of Xenopus laevis was shown to transcribe somatic-type 5S RNA genes -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 -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, TFIHA, was bound both to the actively transcribed somatic-type 5S RNA genes 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.The genome of the frog Xenopus laevis contains two distinct families of genes for 5S RNA, the smallest RNA component of the ribosome. In the developing oocyte of this amphibian, genomic 5S RNA genes of both families are actively transcribed, yielding large amounts of stored 5S RNA for subsequent ribosome production during embryogenesis. The oocyte-type family of 5S RNA genes, with approximately 20,000 copies per haploid genome, is transcribed during oogenesis but is relatively inactive during embryogenesis and is switched off in somatic cells. In contrast, the somatic-type family of 5S RNA genes, with approximately 400 copies per haploid genome, is transcribed during both oogenesis and embryogenesis and is also active in somatic cells. Transcription of Xenopus 5S RNA genes has been extensively studied both in vitro and in vivo, in part because these genes provide a well-defined example of differential regulation of gene expression during development (5,6,16).Like other genes transcribed by RNA polymerase III, such as the tRNA genes, 5S RNA genes require factors TFIIIB and TFIIIC for transcription (26,29). Unlike the tRNA genes, however, transcription of 5S RNA genes requires an additional factor, TFIIIA (8). This protein has been purified to homogeneity and extensively studied (1,11,23). The first step in the kinetic pathway for the transcription of 5S RNA genes involves the binding of TFIIIA to the internal control region within the 120-base-pair SS RNA coding sequence. The mode of DNA binding by TFIIIA is thought to involve multiple Zn2'-stabilized domains of TFIIIA, each of the domains interacting with a specific stretch of DNA approximately 5 base pairs long (21). After formation of a complex between TFIIIA and the 5S RNA gene, the other transcription factors, TFIIIC and TFIIIB, can bind in turn, forming a stable transcription complex which in the presence of RNA polymerase III is capable of directing multiple rounds of transcription of the 5S RNA gene (4, 18, 27). * Corresponding author.Whereas the nucleotide sequences flanking the oocytetype and somatic...

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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.

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