In most species, transcription does not occur immediately upon fertilization. Rather, the initial protein synthesis is based on the translation of mRNAs that are already present within the oocyte. This means that a large number of ribosomes must be present to translate these messages. In many respects, the ribosome can be considered as a differentiated cell product of the oocyte. This is especially so in amphibians, where the oocyte contains around 200,000 times the number of ribosomes found in most somatic cells. For one cell, that number of ribosomes is truly spectacular. In fact, in those zygotes lacking the nucleolar organizing regions that make ribosomal RNA, development can still continue on the stored ribosomes up until the feeding tadpole stage (at which time the organism finally dies from the lack of ribosomes needed for growth).
Synthesis of ribosomal RNA in amphibian oocytes occurs during the prolonged prophase of the first meiotic division. Specifically, rRNA synthesis takes place during the months-long diplotene stage, after pairing and replication of the homologous chromosomes. The "large" rRNA genes of the frog Xenopus laevis are diagrammed in Figure 1. There is a 5' leader sequence followed by the coding sequence for 18S rRNA. This is followed by a transcribed spacer, the gene for the 5.8S rRNA gene, another transcribed spacer, and then the gene for the 28S rRNA. There are no introns within these genes, and the entire unit is transcribed into a 40S rRNA precursor, which is then processed to form the three rRNAs.
Ribosomal Gene Amplification
One of the mechanisms for synthesizing so many ribosomes is the repetition and amplification of the rRNA genes. *Repeated genes denotes inherited multiplicity, wherein a given sequence is present numerous times in the genome of every cell. (The 5S rRNA genes, represented thousands of times per genome, constitutes a set of repeated genes). Amplified genes are those DNA sequences that are reiterated in specific cells only. Their multiplicity is not inherited. The 28S, 18S, and 5.8S rRNA genes are repeated (about 900 times per cell) and amplified (in the oocyte). There are two homologous regions of large-rRNA genes per diploid frog cell. Each region contains roughly 450 copies of the unit described in the preceding section, and the copies are separated within the region by nontranscribed spacers. All the rRNA gene units on a chromosome are identical, but the nontranscribed spacers vary in length from unit to unit, even on the same chromosome. We would expect, then, that the diplotene oocyte (in which the DNA has already replicated) would contain 4 × 450, or roughly 1800, genes coding for rRNA. But apparently this is not enough! Rather than a few thousand copies, one finds nearly a million rRNA genes in the maturing oocyte. One sees not four nucleoli, but thousands (Figure 2). These extra nucleoli reside within the nucleus but are not attached to any chromosome, as nucleoli usually are. When the hybridization of radioactive 28S rRNA to oocyte DNA was compared with hybridization to somatic cell DNA, it was determined that the rRNA genes were amplified 1500 times over their normal amount. If each nucleolus contains 450 gene copies, we would estimate that there are 6.8 × 105 genes for the large-rRNA precursors in the amphibian oocyte (Brown and Dawid 1968). These extra rDNA genes are made during the pachytene stage of meiosis and are transcribed during the diplotene stage. When the oocyte nucleus disintegrates during the first meiotic division, the extra nucleoli are thrown into the cytoplasm and are destroyed.
Visualization of Transcription from Amplified Genes
The amplified ribosomal RNA genes of amphibian oocytes were the first eukaryotic genes to be isolated and purified. They were also the first genes whose transcription could actually be seen by electron microscopy. Knowing that these genes would be intensely active in transcribing rRNA during the diplotene stage of meiosis, Miller and Beatty (1969) used low-ionic-strength buffers to disperse the chromatic strands for electron microscopy. The circles of DNA unwound to reveal the configuration seen in Figure 1. There is an axial core of DNA upon which rRNA is being synthesized. Numerous rRNA chains are being transcribed from any one unit, and several such units are seen in this picture. In each unit, transcription begins at the small end of the "Christmas tree" and continues until the large-rRNA precursor is formed. At each junction of the DNA with an RNA transcript, there is a molecule of RNA polymerase. The length of the RNA in these large transcripts corresponds to about 7200 bases, which is roughly the size estimated for the ribosomal precursor. Between the transcribing units of DNA are the "nontranscribed spacers." These electron micrographs beautifully reveal eukaryotic DNA in the act of transcription.
Regulation of the 5S rRNA Gene
In addition to the large-rRNA transcription unit, there is the transcription unit of the 5S ribosomal RNA. The 5S rRNA is made from another region of the genome, and these 5S genes are present in thousands of copies in the genome (and are not amplified). In each haploid genome, there are 20,000 "oocyte" 5S genes (that are only active in the oocyte) and 400 "somatic" 5S genes (that are functional in every cell). They are all transcribed during early oogenesis. During late oogenesis, all the 5S genes are turned off until the midblastula transition takes place. At midblastula, both oocyte and somatic 5S genes are expressed, but by the end of the blastula stage, only the somatic 5S genes are active (Korn 1982). The RNA polymerase controlling the production of 5S rRNA genes is RNA polymerase III, and it has a unique promoter. Whereas the promoters for RNA polymerases I and II are in the 5' flanking regions of the gene (as they are in bacteria), the promoters for RNA polymerase III are in the center of the gene (Figure 2; Bogenhagen et al. 1980; Sakonju et al. 1980).
As in the case of RNA polymerase I and II, the binding of eukaryotic RNA polymerases to their promoters is mediated by transcription factors. A 38.5-kDa protein binds to the promoter region of the 5S rRNA gene and directs the RNA polymerase III to bind (Ng et al. 1979; Engelke et al. 1980). This protein, TFIIIA (i.e., the first transcription factor for RNA polymerase III) is specific for the 5S rRNA gene (it does not bind to the promoters of other RNA polymerase III-dependent genes) and is itself developmentally regulated. There are 1012 TFIIIA molecules per cell in the oocyte, but only 103 TFIIIA molecules per somatic cell (Ginsburg et al. 1984; Scotto et al. 1989). The binding of TFIIIA to the internal promoter of the 5S rRNA genes is very weak. It is greatly stabilized by the nonspecific factor TFIIIC (Lasser et al. 1983; Pieler et al. 1987). This tightly bound complex binds another factor, TFIIIB, which can bind RNA polymerase III (Wolffe and Brown 1988). Once RNA polymerase III binds to this complex, transcription begins. Like the transcription assemblies discussed in Chapter 10, TFIIIA and TFIIIC are assembly factors, and TFIIIB contains the TATA-binding subunit (even though it does not bind to TATA or any other DNA).
It appears, then, that the type and amount of 5S rRNA depends on the level of TFIIIA in the nucleus. But how is that control effected? Brown and Schlissel (1985) injected cloned oocyte and somatic 5S rRNA genes into oocytes and blastula nuclei and showed that TFIIIA has a much higher (over 200-fold) affinity for the control region of the somatic 5S rRNA genes than it does for the oocyte 5S rRNA genes. Moreover, when purified TFIIIA was injected into late-blastula-stage embryos (in which only the somatic 5S rRNA genes are active), the oocyte 5S genes become activated as well. These observations indicated that at low concentrations of TFIIIA, the only genes binding TFIIIA will be of the somatic type; but at higher levels, the oocyte 5S rRNA genes will also become activated. The basis for this difference in the ability of the 5S genes to be activated by TFIIIA was found to reside in three nucleotides that differed between the oocyte and somatic 5S genes. While these three base substitutions were responsible for changing the stability of the 5S gene-TFIIIA complex, they did not directly alter TFIIIA-promoter binding. Rather, the changes in nucleotides caused the TFIIIA protein to become displaced from the promoter by TFIIIC (Wolffe and Brown 1988).
DNase I "footprinting" has shown that TFIIIA binds to the 5S rRNA gene in three places. TFIIIA is incubated with a 5S RRNA gene that has been radio-labeled at one end (as is done for sequencing). The DNA is then digested with DNase I, a relatively nonspecific DNase. Each strand should be broken (on average) only once. The resulting DNA is placed in a gel and separated by electrophoresis as if it were being sequenced. One obtains the same ladders expected of sequencing gels, where each oligonucleotide is one nucleotide shorter than the one above it. But in those regions where the protein (in this case, TFIIIA) has protected the DNA from DNase I treatment, the corresponding oligonucleotides are absent. Such a pattern for the binding of TFIIIA to the Xenopus oocyte 5S rRNA gene is shown in Figure 3, and it shows the three sites where TFIIIA binds to the internal promoter region (Sakonju and Brown 1982).
rRNA Binding by TFIIIA
TFIIIA is a zinc finger transcription factor with nine zinc fingers. Miller and co-workers (1985) suggest that this arrangement enables the TFIIIA to "hold on" to the gene as the RNA polymerase III moves along. Some of the binding regions maintain the hold on the DNA as others relinquish it. TFIIIA is also an RNA-binding protein, and complexes with the transcribed 5S rRNA in the oocyte cytoplasm. The same zinc fingers that bind to the central promoter region of the DNA enable it to bind to the RNA (Theunissen et al. 1992). In Xenopus, the 5S rRNA appears before the large-rRNA transcript. When it enters the cytoplasm, it becomes complexed with TFIIIA. However, when the large transcript is being synthesized and the large ribosomal subunit is being made in the germinal vesicle, the TFIIIA appears to be exchanged for ribosomal protein L5, and the 5S rRNA-L5 complex shuttles back into the germinal vesicle, where it will become incorporated into the 60S ribosomal subunit (Allison et al. 1991).
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