In vitro 5S RNA Synthesis
What is required to reproduce in vitro the regulation of 5S RNA synthesis that is seen in vivo? A conservative approach in attempting to answer this type of question is to begin as near to the biological situation as possible and to work backward towards a system containing only known and purified components, each performing a well-understood function. During each of the steps in moving from the native biological system to the completely defined simple system, one’s criterion of a properly working system is that correct regulation be maintained.
A logical first step in reproducing the biological regulation of Xenopus 5S RNA synthesis is to try injecting DNA coding for 5S RNA into oocytes. Such injections are of medium technical difficulty. Due to the large size of later stage oocytes, they can be isolated free of ovarian tissue, injected, incubated, and synthesis products from individual or pooled oocytes analyzed. Gurdon and Brown found that microinjecting the nuclei of oocytes with either DNA purified from frog erythrocytes or with pure plasmid DNA containing one or more 5S genes yielded synthesis of 5S RNA. This synthesis could continue many hours or even for days after the injection.
The next step toward a defined system also determined whether the “living” oocyte possessed any mystical properties. That is, could 5S RNA synthesis be achieved by an extract of nuclei purified from oocytes, or were intact oocytes required? The experiments showed that nuclei were capable of synthesizing the RNA. A major drawback to the experiments, however, was the labor required to obtain the oocyte nuclei from the ovaries. Not surprisingly, then, a simple cell extract of the entire oocyte was tried, and found to be almost as effective in synthesizing 5S RNA as the nuclear extracts. Generally chromatin, that is DNA plus associ-ated proteins and histones, that is obtained by gently lysing cells and taking whatever sediments with DNA, is a good template DNA.
Oocyte nuclear extracts using either DNA or chromatin were active in synthesizing 5S RNA. When RNA polymerase III was tried instead of the extracts, polymerase plus DNA was inactive, but polymerase with chromatin as a template was active. These experiments demonstrate that at least one factor, presumably a protein, in addition to DNA and RNA polymerase III, is necessary for 5S RNA synthesis and that the factor is associated with chromatin. The question then became one of determining how many factors were required for synthesis of 5S RNA, how many of the factors were associated with chromatin, and whether properties of these factors explained either the shutoff of oocyte and somatic 5S RNA synthesis in the oocyte or the later resumption of only somatic 5S RNA synthesis.
Extracts prepared from unfertilized eggs do not support the synthesis of either type of 5S RNA from a pure DNA template. If these extracts are supplemented with extracts prepared from oocytes that were ac-tively synthesizing 5S RNA, they do synthesize 5S RNA. Thus the unfertilized eggs provide an assay and the immature oocytes provide a source for one of the required factors. Through this sort of approach three factors have been found that stimulate synthesis of 5S RNA TFIIIA, TFIIIB, and TFIIIC. Other genes that are transcribed by RNA polymerase III, like some tRNA genes, the gene encoding the U6 RNA which is involved in splicing, and the adenovirus VA gene, utilize TFIIIB and TFIIIC only. TFIIIA was the easiest to purify and study, and therefore the most is known about it. This protein is monomeric and has a molecular weight 37,000.