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

Abstract

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...

Changing directions in Pol III transcription.

“…However, everything else does not appear to be equal, for in a transcription system derived from mature oocytes, the oocyte 5S RNA genes are transcribed in vitro at only a few percent of the rate of the somatic genes. These results confirm similar results recently reported by Peck et al (1987) and by Millstein et al (1987).…”

Two TFIIIA activities regulate expression of the Xenopus 5S RNA gene families.

“…The so matic-type gene preference observed in oocyte extracts does not depend on levels of TFIIIA either, because suffi cient quantities of this protein are available to bind both active somatic-type and inactive oocyte-type genes Peck et al 1987). We examined several different types of extracts prepared from Xenopus oocytes, eggs, and embryos for their relative ability to transcribe the oocyte-and somatic-type 58 genes (Mill-Stein et al 1987;Peck et al 1987). We found two general classes of extracts: those that give approximately equiv alent levels of transcripts from the two genes (in inde pendent reactions), and those that preferentially support somatic-type transcription.…”

“…Two explanations for preferential transcription of the somatic-type genes in the mature oocyte S-150 might be that these extracts are deficient in some component spe cifically required for oocyte-type transcription or that these extracts may contain a specific repressor of oo cyte-type transcription (Peck et al 1987). In this regard, Millstein et al (1987) have shown that stable inactive complexes are formed on the oocyte-type genes in S-150 extracts and that these complexes are refractory to acti vation after microinjection into living oocyte nuclei; however, Wolffe (1988) has shown that the addition of a crude protein fraction containing TFIIIC and other pro teins to S-150 extracts can elevate oocyte-type transcrip tion.…”

“…Recently, we Peck et al 1987) and others (McConkey and Bogenhagen 1987;Wolffe 1988;Xing and Worcel 1989) have been able to repro duce the preferential transcription of somatic-type genes in vitro with extracts prepared from mature oocytes or eggs (Glikin et al 1984). These results were not depen dent on transcription reaction conditions, DNA concen tration effects, or differential RNA stability.…”

“…These results were not depen dent on transcription reaction conditions, DNA concen tration effects, or differential RNA stability. The so matic-type gene preference observed in oocyte extracts does not depend on levels of TFIIIA either, because suffi cient quantities of this protein are available to bind both active somatic-type and inactive oocyte-type genes Peck et al 1987). We examined several different types of extracts prepared from Xenopus oocytes, eggs, and embryos for their relative ability to transcribe the oocyte-and somatic-type 58 genes (Mill-Stein et al 1987;Peck et al 1987).…”