Cell Nucleus Biogenesis, Structure and Function
405
Fig. 5
Nuclear compartments and transcription.
HeLa cells growing on glass were permeabilized in
an isotonic buffer and transcription performed for
15 min in the presence of BrUTP. Samples were
Fxed and immuno-labeled for Br-RNA containing
sites (right) using a red fluorescence dye and sites
containing the auto-immune antigen Sm (left) using
a green fluorescent dye. The two colors were
recorded in a single 700 nm section of the nucleus
using a laser scanning confocal
microscope-gray-scale images of the two channels
are shown. Note the classical ‘‘speckled’’
distribution of the Sm protein and the complexity of
the transcription sites; the two areas that do not
stain with Sm are nucleoli. The bar is 5
µ
m. See
Pombo, A., Cook, P.R. (1996) The localization of
sites containing nascent RNA and splicing factors,
Exp. Cell Res.
229
, 201–203 for details. Published
with permission of Academic Press.
Sm
Br-RNA
are polymerized, processed and assembled
into the required mRNA–protein complex
before being released for export to the cy-
toplasm. For a typical transcript, events
occurring at the transcription site take
roughly 15 min to complete.
A likely corollary of this arrangement
is that the transcription centers form
a functional nuclear compartment that
is spatially structured by an association
with a component of the interchromatin
compartments – perhaps a nucleoskele-
ton. Hence, during transcription the chro-
matin loops must be locally dynamic as
RNA synthesis forces the gene to progres-
sively associate (i.e. from promoter to 3
0
end) with the synthetic center. The idea
that genes might be associated with the ac-
tive center during synthesis and displaced
from it thereafter de±nes a type of spatially
determined ‘‘transcription cycle’’ within
which the interplay between chromatin
dynamics and promoter bound factors will
dictate levels of gene expression. The dy-
namic behavior of transcription factors
will also be central to this control. In-
terestingly, some classes of transcription
factor can be shown to associate with
chromatin only transiently, whereas others
are more stable, and might even remain
bound to chromatin throughout mitosis.
Fac
torssuchasTBP(TATAb
ind
ingpro
-
tein), which fall in the second category,
are ideal candidates to mark active genes
and regulate the association of these genes
with transcription sites.
Replication factories
DNA replication pro-
vides an interesting insight into how
nuclear structure and function can be
linked. We will see that DNA foci pro-
vide the best candidates for any structural
units of chromosome structure (below).
However, it is also important to recognize
that the organization of these structures
might be central to both the replication
process and how the replication program
proceeds. DNA foci contain small groups
of replicons, which operate as spatially sta-
ble replicon clusters and provide targets for
the assembly of replication factories. Like
transcription factories, these arise through
DNA–protein and protein–protein inter-
actions that generate nuclear sites in which
DNA synthesis can be performed with
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