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
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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