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Chapter: Genetics and Molecular Biology: Xenopus 5S RNA Synthesis

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Switching from Oocyte to Somatic 5S Synthesis

In vitro transcription experiments revealed an unexpected property of5S transcription.

Switching from Oocyte to Somatic 5S Synthesis

In vitro transcription experiments revealed an unexpected property of5S transcription. Brown and co-workers discovered that once TFIIIA had bound to a DNA molecule, it did not easily or rapidly dissociate and bind to a different molecule. Even multiple rounds of transcription could not displace a bound TFIIIA molecule. Demonstrating this prop-erty is rather simple. Consider two different kinds of DNA templates for 5S RNA, one containing an insertion of a few bases so that the resulting product is distinguishably larger when run on polyacrylamide gels. When the two types of templates were added to a reaction together, both sizes of RNA were synthesized, but under conditions of limiting TFIIIA; whichever template was first added encoded all the resulting RNA. The TFIIIA remained associated with its DNA upon dilution or even through 40 rounds of transcription.

Complexes of TFIIIA formed in vivo also possess high stability. These experiments can be done in the following way. As increasing quantities of DNA containing the 5S gene are injected into oocytes, eventually the synthesis of 5S RNA saturates. This can be shown to be due to the titration of TFIIIA factor. Injection of TFIIIA factor relieves the satura-tion and permits synthesis of still more 5S RNA. Secondly, if template for tRNA, which does not require TFIIIA, is included in the second injection of 5S template along with template for 5S RNA, only the tRNA template is active. This shows that RNAP III has not been titrated out. Once it is known that TFIIIA is limiting, the inactivity of the 5S template added to an already template saturated oocyte means that no significant amount of TFIIIA dissociates from the first template during the experi-ment.

The somatic 5S genes, but not the oocyte 5S genes in chromatin isolated from somatic cells are active in some types of extracts. What maintains the set state of these two types of genes? Not surprisingly, the somatic genes contain bound TFIIIA. Not only do the oocyte genes lack TFIIIA, but provision of extra TFIIIA does not activate them. One explanation could be that histones on the DNA block access of TFIIIA to its binding site.

 Indeed, various histones can be stripped from chromatin by extraction in buffers containing high concentrations of salt. Extraction with 0.6 M NaCl removes histone H1 and at the same time permits TFIIIA activation of oocyte 5S genes. Similarly, stripping the chromatin of histones by high salt, addition of TFIIIA, and then reformation of chromatin by addition of the histones and slow removal of the salt leave the oocyte genes active.

We can generate a simple picture to explain the histone results. After replication, the DNA is bare behind the replication fork. Under these conditions both TFIIIA and histones should be able to bind to the 5S gene. If TFIIIA binds first, a complete transcription complex forms. This possesses sufficient stability to remain in place for long periods. Once it has the complex is present, nucleosomes are free to form on either side. The presence of the TFIIIA and the absence of histone bound at the 5S gene means that transcription can begin despite the presence of histones elsewhere on the template. Conversely, if histones bind first to the gene, they remain bound and prevent formation of a transcription complex. In this case the 5S gene cannot be activated, even by the subsequent addition of large amounts of TFIIIA. Hence the long-term transcriptional abilities of the 5S genes could be determined at the time of their replication.

Just as a transcription complex must be present to prevent histones from binding to the critical part of the 5S gene, those genes transcribed by RNAP II must also be prevented from being locked in an off state. General or specific transcription factors must perform this role for mRNA genes. One attractive candidate for a general transcription factor that would often serve this role is TFIID, which binds to the TATA box of the promoter region of genes transcribed by RNA Pol II.

 

The high stability of TFIIIA binding raises the possibility of a simple explanation for the switch between oocyte and somatic 5S RNA synthe-sis. In principle, the developmental switch could be simply accom-plished if TFIIIA itself had a substantial binding preference for somatic genes. Then, in the developing oocyte with its vast excess of TFIIIA, both somatic and oocyte genes would be active. However, as TFIIIA is titrated out into the 7S and 42S particles, the functional excess would vanish and the genes would be occupied by TFIIIA in proportion to the protein’s binding preference for the two slightly different sequences. The main problem with this explanation is that, experimentally, TFIIIA has too small a preference for the somatic genes to explain the thousand-fold change in the relative activities of the two types of genes. If the oocyte sequence of the 5S gene itself is changed nucleotide by nucleotide to that of the somatic 5S, there is some effect in the correct direction. The changes at positions 47, 53, 55, and 56 of the gene do make the oocyte gene behave more like the somatic genes, in oocyte extracts in which the activity of TFIIIA is being monitored, but the change is insufficient to explain the dramatic difference between the two genes in vivo. The conclusion has to be that the nucleotide differences between the somatic and oocyte 5S RNA genes are sensed by the entire transcription complex consisting of TFIIIA, B, C, and RNA polymerase III.


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