Download nuclear structure (2): the nucleolus

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NUCLEAR STRUCTURE (2): THE NUCLEOLUS
Reading: pp. 538-547; p. 95 (Svedberg units)
More than 95% of the RNA in a eukaryotic cell is rhe RNA that forms part of the structure
of ribosomes. This is the ribosomal RNA (rRNA). The “size” of macromolecules such as
proteins and RNA can be determined by how rapidly the molecule moves to the bottom of the
centrifuge tube during ultracentrifugation. Actually the “size” determined by this method is a
reflection of both the mass and the shape of the molecule. The units used are Svedberg units
(S). The larger the S value, the larger the molecule. But S values do not add up in a linear
fashion because of the influence of both mass and shape on the detrmination of the S value.
The important facts in the previous diagram are:
(1) A functional ribosome consists of a large subunit and a small subunit.
(2) Together, the two subunits possess about 80 different ribosomal proteins.
(3) The large subunit contains three ribosomal RNA molecules. The sizes of the
ribosomal RNAs are 5S, 5.8S, and 28S.
(4) The small subunit contains one ribosomal RNA that is 18S in size.
The ribosomal RNA (rRNA) has two functions;
(1) It serves as the skeleton for the attachment of the ribosomal proteins.
(2) It is actually the rRNA that catalyzes the formation of the peptide bonds.
The ribosomal proteins give the required shapes to the ribosomal subunits to allow the proper
binding of the transfer RNA molecules, mRNA, and exit of the growing polypeptide.
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The ribosomal subunits are synthesized in nucleoli.
Some of the events related to the synthesis of the ribosomal subunits that occur in the nucleolus
are:
(1) Transcription of the ribosomal DNA genes (rDNA genes) that occur in the
nucleolus.The transcription is catalyzed by RNA polynerase I (RNA pol I).
(2) The RNA molecule that results from this transcription has a size of 45S. It is cut to
form three of the four ribosomal RNA (rRNA) molecules that are rquired for the
ribosomal subunits.
(3) Proteins are added to the nascent RNA transcript. These proteins include: ribosomal
proteins that will remain associated with the rRNA; proteins (enzymes) that cut the
RNA transcript to form the 5.8S, 18S, and 28S ribosomal RNA molecules; and proteins
that help the assembly of the ribosomal subunits (such as nucleolin).
(4) The 5S ribosomal RNA is transcribed from a gene located outside the nucleolus. This
ribosomal RNA must enter the nucleolus so it can be assembled into the large subunit.
The processing of the 45S ribosomal RNA transcript.
There are about 400 copies of the gene for the 45S rRNA transcript in each human somatic
cell. The reasons for the large number of these genes are:
(1) So many ribosomes must be made.
(2) The only possible amplification step in the making of RNA is the possibilty of more
than RNA polymerase molecule transcribing the gene at a given time. (For proteins
there is the second amplification possibilty of having multiple ribosomes on a
messneger RNA (mRNA) molecule.)
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Synthesis of rRNA in the nucleolus
COMPLETED 45S
rRNA TRANSCRIPT
DIRECTION OF TRANSCRIPTION
PROMOTER
SEQUENCE
5.8S, 18S AND
28S rRNA
CUTTING
RNA
POLYMERASE 1
TERMINATION SEQUENCE ON
THE TEMPLATE DNA STRAND
TRANSCRIPTION UNIT FOR
THE 45S rRNA TRANSCRIPT
In this case the amplication steps are:
(1) Multiple RNA polymerase molecules on the transcription unit (the 45S rRNA gene),
transcribing simultaneously.
(2) Multiple copies (400 per human somatic cell) of the 45S rRNA gene.
In amphibians, which may large eggs with a lot of cytoplasm containing a lot of ribosomes,
extrachromosomal nucleoli can be formed. To from these the nucleolar organizing regions
(NORs) (chromosomal regions containg the 45S rRNA genes) are replicated (DNA replication)
a hundred times or more. These NORs become free of the chromosomes and each one then
forms a small extrachromosomal nucleolus.
This is a light microscope view of
anuclues isolated from an amphibian
oocyte (Xenopus, the clawed toad).
The hundreds of extrachromosomal
nucleoli can ce seen. Formation of
such large numbers of nucleoli is
clearly another from of
“amplificatio” in terms of making
ribosomes more rapid;y.
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Synthesis of most proteins
DIRECTION OF TRANSCRIPTION
pre-mRNA TRANSCRIPT
PROMOTER
SEQUENCE
mRNA
SPLICING OUT
OF INTRONS
RNA POLYMERASE 1
RNA POLYMERASE II
GENE
DIRECTION OF TRANSLATION
PROTEIN
mRNA
MULTIPLE RIBOSMES ON
ONE mRNA MOLECULE
(CALLED A POLYSOME)
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DIRECTION OF TRANSCRIPTION
The top panel shows a nucleolar spread as seen in the electron microscope. Note how many
“christmas trees” there are. Each “tree” is a 45S RNA gene being rapidly transcibed. Ans this
is only a small sample of the entire nucleolar spread! Notice that there a a non-transcribed
region between each “tree” (each 45S RNA gene).
The lower panel shows a higher magnification of two of the 45S rRNA genes. Note the
following;
(1) The promoter is located at the upper left hand end of the genes.
(2) The black “dots” on the DNA (at the bottom of each “branch”) are the RNA
polymerase molecules.
(3) The “branches” are the nascent 45S rRNA molecules.
(4) At various locations along each “branch” (each nascent 45S rRNA molecule) are
black dots. These are places where proteins have bound.
(5) The nascent RNA molecules do not appear to be as long as the DNA template on
which they transcribed. Of course they have to be the same length before any
processing occurs. The reason they do not appear to be the same length is that the
proteins that have already been added cause the RNA transcript to “bunch up”.
(6) The black dots in the non-transcibed spacer region are not RNA polymerase
molecules. Instead they are nucleosomes (the two tures of DNA double hexix
wrapped around 8 histone molecules.
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DIRECTION OF TRANSCRIPTION
The “trees” are diagrammatic, not actual genes being transcribed as are shown in the
previous figure. Note the following;
(1) There are two types of “spacer” in in the DNA template. One spacer is a length of
DNA that is not transcribed and which separtes the individual copies of the 45S
rRNA gene. Not surprisingly, these are called the non-transcribed spacers.
Then there are the spacers (yellow bars) that separate the DNA sequences that
actually have the information for the synthesis of the 5.8S, 18S, and 28S rRNA
molecules. These are spacers are actually transcribed to form the 45S RNA
transcript. These transcribed spacers must then be cut out of the 45S RNA
trnascript.
(2) Each gene has a promoter and a small sequence at the downstrean end that
signals “termination” for the RNA polymerase molecules.
18S
5.8S
28S
NON-TRANSCRIBED
SPACER
NON-TRANSCRIBED
SPACER
THE 45S DNA TRANSCRIPTION UNIT (TEMPLATE)
TRANSCRIPTION
CUTTING OUT OF THE
TRANSCRIBED SPACERS
18S rRNA
5.8S rRNA
28S rRNA
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The nucleolar organizing region (NOR)
In the above review diagram of the role of the nucleolus in the synthesis of the ribosomal
subunits you can see the term “loop of nucleolar organizer DNA”. This is really just another
term for “all the 45S rRNA genes and the non-transcibed spacer DNA”. This is usually called
the nucleolar organizing region (NOR). A nucleolus can form at each of these regions, and in
human somatic cells just after their division this is the case. In fact, 10 nucleoli can be
observed in these cells just after division. In humans, the 10 NORs are located on 5 separate
homologous pairs of chromosomes (for a total of 10 seoarte chromosomes!). later in the life
cycle of the cells these individual nucleoli usually coalesce to from large nucleolus (see the
diagram below).
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This diagram shows the appearance of the nucleolus (nucleoli) during the life
cycle of somatic cell capable of cell division.
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Key words (inluding for previous set of notes)
transcription factory
replication factory
painting chromosomes
nuclear lamina
intermediate filaments
nuclear envelope
inner nuclear membrane
outer nuclear membrane
heterochromatin
euchromatin
chromatin
telomer
fluorescence in situ hybridization (FISH)
biotin
biotinylation of a deoxyribonucleotide
avidin
fluorophore covalently bound to avidin
separation of DNA strands (“melting”)
RNA polymerase
DNA polymerase
nucleolus
nucleolar organizing region
45S ribosomal RNA genes
45S ribosomal RNA transcript
transcribed spacer
non-transcribed spacer
processing (cutting) of 45S RNA transcript
about 80 ribosomal proteins
45S rRNA
5.8S rRNA + 18S rRNA + 28S rRNA
5S ribosomal RNA genes (extranucleolar)
fibrous region of nucleolus (rDNA + 45S rRNA transcript)
granular region of nucleolus (developing large and small ribosomal subunits0
extrachromosomal nucleoli of amphibians
“amplification steps” for RNA and protein synthesis
polysome ( = polyribosome)
small ribosomal subunit (40S)
large ribosomal subunit (60S)
Svedberg unit (by ultracentrifugation)
ribonulceoprotein particle (specific proteins bound to specfic RNA molecules)
nucleosome
histone