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Lecture 5

Molecular Cell Biology Lecture 5.docx


Department
Biological Sciences
Course Code
BIOB11H3
Professor
Dan Riggs
Lecture
5

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Molecular Cell Biology Lecture 5
1. Eukaryotic mRNAs are synthesized as larger precursors known as hnRNAs.
2. Pulse chase experiments are useful for determining how this occurs; involves labeling
macromolecules with radioactive NTPs, separating RNAs by size (by centrifugation and
fractionation: absorbance & radioactivity measurements follow).
3. Polymerase II is a large multisubunit complex, which binds upstream of the gene to be transcribed.
Changes in the complex (addition or loss of subunits, phosphorylation of subunits) alter its activity
such that initiation, elongation and termination occur properly.
4. A mature mRNA usually consists of a 5’ cap, the 5’ untranslated leader, the ‘coding’ region, the 3’
untranslated region, and a poly A tail.
5. Removal of introns occurs by RNA splicing reactions. Splicing can be autonomous (in the case of
self-splicing RNAs), or mediated by a multisubunit complex known as the spliceosome.
6. Consensus sequences at the intron/exon junctions are used to identify the proper splice sites.
7. Hybridization assays and DNA sequencing are useful for determining exactly what has occurred in
mRNA maturation.
o When a eukaryotic cells are incubated for a short period in [3H]uridine or [32P]phosphate and
immediately killed, most of the radioactivity is incorporated into a large group of RNA molecules that
have the following properties:
a. They have large molecular weights (up to about 80S, or 50,000 nucleotides);
b. As a group, they are represented by RNAs of diverse (heterogeneous) nucleotide sequence;
c. They are found only in the nucleus.
Because of these properties, these RNAs are referred to as heterogeneous nuclear
RNAs (hnRNAs), and the red radioactivity line in Figure 11.17a indicates them.
When cells that have been incubated in [3H]uridine or [32P]phosphate for a brief pulse
are placed into unlabeled medium, chased for several hours be- fore they are killed and
the RNA extracted, the amount of radioactivity in the large nuclear RNAs drops sharply
and appears instead in much smaller mRNAs found in the cytoplasm (red line of Figure
11.17b). I. Label cells for short period of time with 32P
II. Purify RNA, centrifuge to separate molecules by
size
III. Determine OD (RNA) and radioactivity (labeled
hnRNA RNA).
Large molecules come out first followed by the smaller
molecules
The blue lines indicate the optical density (absorbance of UV
light) of each fraction, provide information about the amount
of RNA in each fraction following centrifugtation = evident
that most RNA in cell is present as 18S and 28S rRNA
The red lines indicate radioactivity in each fraction, provide
information about the number of radioactive nucleotides
incorporated into different-sized RNAs during the brief pulse.
Evident that neither hnRNA nor the mRNA consititute
significant fraction of the RNA reason is that these
RNAs are degraded after relatively brief period of
time.
Majority of RNA synthesized is hRNA but about 90%
of the total RNA of a cell is rRNA
o Something to keep in mind for figures 11-13 and 11-17: Most of newly synthesized RNA in the cell is
hnRNA..BUT, note that..Most of the stable RNA of the cell is rRNA (over 90%)
Half-life how long before degradation or destroyed on purpose
Half-life of hnRNA/mRNA:minutes to hours (average 40min)

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Half-life of rRNAs: days/months; accumulation occurs
o For rRNA (pol I): initial 45S 28S, 18S, 5.8S. What about the mRNAs? Processing events? YES
a. Pulse chase strategy used (fig 11-17)
Conclusion: mRNAs are synthesized as precursors
hnRNA=heterogenous nuclear RNA
~30,000 active genes-many transcripts of various sizes
o All eukaryotic mRNA precursors are synthesized by RNA polymerase II, an enzyme composed of a
dozen different sub-units that is remarkably conserved from yeast to mammals. Transcription of a pol
II gene is regulated by:
A. Transcription factors which recruit RNA pol.
B. Sequences in the promoter of the gene
Binding sites for transcription factors and binding sites for pol II subunits
RNA polymerase II binds the promoter with the cooperation of a number of general
transcription factors (GTFs) to form a preinitiation complex (PIC).
The promoter elements that nucleate PIC assembly lie to the 5 side of each
transcription unit, although the enzyme makes contacts with the DNA on both sides of
the transcription start site
A critical portion of the promoter of such genes lies be- tween 24 and 32 bases upstream from
the site at which transcription is initiated (Figure 11.18a). This region often contains a
consensus sequence that is either identical or very similar to the oligonucleotide 5-TATAAA-
3 and is known as the TATA box.
The first step in assembly of the preinitiation complex is binding of a protein, called
TATA-binding protein (TBP), recognizes the TATA box of these promoters (Figure
11.18b). Thus, as in bacterial cells, a purified eukaryotic polymerase is not able to
recognize a promoter directly and cannot initiate accurate transcription on its own.
The “TATA” box – in eukaryotes is located about -25 relative to the
transcription start site
While TBP binds TATA, other subunits of the TFIID complex bind to other regions of the DNA,
including elements that lie down- stream of the transcription start site.
Binding of TFIID sets the stage for the assembly of the complete preinitiation complex, which is
thought to occur in a stepwise manner as depicted in Figure 11.18b.
TBP = TATA binding protein bends DNA, unwinding occurs
Other TFs recruited by TBP/TAF
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