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Mrs. Ofelia Solano Saludar: Department of Natural Sciences University of St. La Salle Bacolod City
Mrs. Ofelia Solano Saludar: Department of Natural Sciences University of St. La Salle Bacolod City
(5) After an
activation step
requiring ATP-
dependent
phosphorylation of
the RNA
polymerase
molecule, the
polymerase can
initiate
transcription at the
startpoint.
The TATA-binding protein (TBP) is a subunit of the TFIID
and plays a role in the activity of both RNA polymerase I and
III transcription.
TBP is also essential for transcription of TATA-less genes.
TBP differs from most DNA-binding proteins in that it
interacts with the minor groove of DNA, rather than the major
groove and imparts a sharp bend to the DNA.
TBP has been highly conserved during evolution.
When TBP is bound to DNA, other transcription-factor
proteins can interact with the convex surface of the TBP
saddle.
TBP is required for transcription initiation on all types of
eukaryotic promoters.
Transcription of eukaryotic pre-mRNAs often proceeds beyond the 3’ end of the mature
mRNA. An AAUAAA sequence located slightly upstream from the proper 3’ end then
signals that the RNA chain should be cleaved about 10-35 nucleotides downstream from
the signal site, followed by addition of a poly-A tail catalyzed by poly(A) polymerase.
To give the mRNA
stability, a 5’ “cap” (a
guanosine nucleotide
methylated at the 7th
position) is joined to the
1st nucleotide in an
unusual 5’ -5’ linkage.
During the capping
process, the first two
nucleotides of the
message may also
become methylated.
In addition to the 5’ cap and poly-A tail, mRNA in
eukaryotes is first made as heterogeneous nuclear
mRNA (or pre-mRNA), and then processed into mature
mRNA through the splicing out of introns.
Restriction
enzyme
analysis has
revealed the
presence of
introns in
eukaryotic
DNA.
Hybridization of a
eukaryotic mRNA
molecule with a gene
which has one intron
will produce two
single-stranded DNA
loops where the
mRNA has
hybridized to the
DNA template strand
plus an obvious
double-stranded DNA
loop. The double-
stranded DNA loop
represents the intron,
which contains
sequences that do not
appear in the final
mRNA.
Alternative splicing results in alternate forms of mRNA
and proteins.
Distinct isoforms of individual domains of multidomain proteins found in
higher eukaryotes often are expressed in specific cell types as the result of
alternative splicing of exons
The ≈75-kb fibronectin gene (top) contains multiple exons. The EIIIB
and EIIIA exons (green) encode binding domains for specific proteins on
the surface of fibroblasts. The fibronectin mRNA produced in fibroblasts
includes the EIIIA and EIIIB exons, whereas these exons are spliced out
of fibronectin mRNA in hepatocytes. In this diagram, introns (black lines)
are not drawn to scale; most of them are much longer than any of the
exons.
Spliceosomes remove introns from pre-mRNA. The spliceosome
is an RNA-protein complex that splices intron-containing pre-
mRNA in the eukaryotic nucleus.
In a stepwise
fashion, the pre-
mRNA assembles
with the U1 snRNP,
U2 snRNP, and
U4/U6 and U5
snRNPs (along with
some non-snRNP
splicing factors),
forming a mature
spliceosome.
The pre-mRNA is then
cleaved at the 5’ splice
site and the newly
released 5’ end is linked
to an adenine (A)
nucleotide located at the
branch-point sequence,
creating a looped lariat
structure. Next the 3’
splice site is cleaved and
the two ends of the exon
are joined together,
releasing the intron for
subsequent degradation.
TERMINATION
In many of the genes transcribed by RNA polymerase II,
transcription can end at multiple sites located within a span
of hundreds or thousands of base pairs.
Termination is coupled to cleavage, which is carried out by
a termination factor that associates with RNA polymerase I
and III.
This complex may suppress termination until the consensus
sequence that marks the cleavage site is encountered.
mRNA is cleaved by the complex 10 to 35 base-pairs
downstream of a AAUAAA sequence (which acts as a poly-
A tail addition signal).
Unlike rho, which binds to the newly transcribed
RNA molecule, the termination factor for RNA
polymerase I binds to a DNA sequence downstream
of the termination site.
RNA polymerase III transcribes a terminator
sequence that produces a string of U’s in the RNA
molecule, like that produced by the rho-independent
terminators of bacteria.
Unlike rho-independent terminators in bacterial cells,
RNA polymerase III does not require that a hairpin
structure precede the string of U’s.
SUMMARY: EUKARYOTIC TRANSCRIPTION
Several types of DNA sequences take part in the initiation of
transcription in eukaryotic cells. These sequences generally serve
as the binding sites for proteins that interact with RNA polymerase
and influence the initiation of transcription.
Sequences that affect transcription, called promoters, are adjacent
to or within the RNA coding region and are relatively fixed with
regard to the start site of transcription. Promoters consist of a core
promoter located adjacent to the gene and a regulatory promoter
located farther upstream.
Other sequences, called enhancers, are distant from the gene and
function independently of position and direction. Enhancers
stimulate transcription.
General transcription factors bind to the core promoter near the
start site and, with RNA polymerase, assemble into a basal
transcription apparatus.
The TATA-binding protein (TBP) is a critical transcription factor
that positions the active site of RNA polymerase over the start
site.
Transcriptional activator proteins bind to sequences in the
regulatory promoter and enhancers and affect transcription by
interacting with the basal transcription apparatus.
Proteins binding to enhancers interact with the basal transcription
apparatus by causing the DNA between the promoter and the
enhancer to loop out, bringing the enhancer into close proximity
to the promoter.
The three RNA polymerases found in eukaryotic cells use
different mechanisms of termination.
EUKARYOTIC RNA POLYMERASES
RNA polymerase I promoters have two key components: (1) the core element, which
surrounds the start site and is sufficient to initiate transcription, and (2) the upstream
control sequence, which increases the efficiency of the core promoter. The basal
transcription apparatus assembles at RNA polymerase I promoters.
RNA polymerase III
recognizes several
different types of
promoters. OCT and
PSE are
consensus sequences
that may also be
present in RNA
polymerase II
promoters.
Ribosomal RNA processing involves cleavage of multiple
rRNAs from a common precursor.
The eukaryotic transcription unit that includes the genes for the
three largest rRNAs occurs in multiple copies and arranged in
tandem arrays with non-transcribed spacers separate the units.
Each transcription unit includes the genes for the three rRNAs
and transcribed spacer regions.
The transcription unit is transcribed by RNA polymerase I into
a single long transcript (pre-rRNA) with a sedimentation
coefficient of about 45S.
RNA processing yields mature rRNA molecules.
RNA cleavage actually occurs in a series of steps which varies
in order with the species and cell type but the final products are
always the same three types of rRNA molecules.
Every tRNA gene is transcribed as a precursor that must be
processed into a mature tRNA molecule by the removal,
addition and chemical modification of nucleotides.
Processing for some tRNA involves:
o removal of the leader sequence at the 5’ end
o replacement of two nucleotides at the 3’ end by the
sequence CCA (with which all mature tRNA molecules
terminate)
o chemical modification of certain bases
o excision of an intron
The mature tRNA is often diagrammed as a flattened cloverleaf
which clearly shows the base pairing between self-
complementary stretches in the molecule.
Long double-stranded RNAs (dsRNA) Upon introduction, the
occur naturally in cells. long dsRNAs with
complementary sequence
of a part of the target
gene, enter a cellular
pathway that is commonly
referred to as the RNA
interference (RNAi)
pathway
The dsRNAs get
processed into 20-25
nucleotide small
interfering RNAs
(siRNAs) by an RNase
III-like enzyme called
Dicer.
The siRNAs assemble into
endoribonuclease containing
complexes known as RNA-
induced silencing complexes
(RISCs), unwinding in the
process.
Activated RISC then binds to
complementary transcript by
base pairing interactions
between the siRNA antisense
strand and the mRNA.
The bound mRNA is cleaved
and sequence specific
degradation of mRNA results
in gene silencing.
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MicroRNAs ("miRNAs")
are single-stranded RNA
molecules containing about 22
nucleotides and thus about the
same size as siRNAs.
These are generated by the
cleavage of larger precursors
using Dicer.
They function as post-
transcriptional regulators of
gene expression.
They act by either destroying
or inhibiting translation of
several mRNAs, usually by
binding to a region of
complementary sequence in
the 3'-UTR region of the
mRNA.
http://www.nature.com/ng/supplements/micrornas/video.html