Transcription regulation in Eukaryotes

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Transcription regulation in Eukaryotes
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Chapter 6 Eukaryotic RNA Polymerase and general
transcription factors -Eukaryotic RNA
Polymerases -General Tr. Factors and Initiation
of Tr. by RNA Pol.II -Transcription by RNA Pol I
an II Regulation of transcription in
eukaryotes -Cis-acting regulatory
sequences -Promoters and enhancers -Transcriptiona
l regulatory proteins -Structure and function of
transcriptional activators -Eukaryotic
repressors -Relationship of chromatin structure
to transcription -DNA Methylation RNA Processing
and turnover -Processing of ribosomal and
transfer RNAs -Processing of mRNA in
Eukaryotes -Splicing Mechanisms -Alternative
Splicing -RNA Editing -RNA Degradation
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RNA polymerases -II (protein-coding genes)
-I and III (rRNA
and tRNA)
-Mitochondria and chloroplasts (similar
to bacterial RNA
Polymerases) Eukaryotic RNA polymerases share
some common subunits and also the need to
interact with other proteins to
initiate transcription. These helper proteins
are called TRANSCRIPTION FACTORS
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RNA polymerase II holoenzyme its the functional
transcription complex including RNA polymeraseII,
TFIIB, E,F,H, plus other proteins. It can bind to
a promoter via TFIID (TBPTAFs) association.
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RNA polymerase I and III The TATA-binding
protein TBP, is required for initiation of
transcription by all three polymerases. RNA
polymerase I for ribosomal RNA genes 45S-pre
mRNA

28S, 18S, 5.8S rRNA
RNA polymerase III for tRNAs and 5S rRNA. Their
promoter lies within of
the transcribed sequence.
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mRNA
5
3
1
CAT
TATA
GCGCG
enhancer
TATA
GCGC
CAT
-25
-50
-75
3
5
ATG
promoter
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Cis-acting Regulatory Sequences (Promoters and
enhancers) Frequently located upstream the TATA
box. CCAAT (CAT box) GGGCGG (GC box)
proximal to the TATA Enhancers located further
upstream of the transcription site.
They bind to transcription factors that
regulate RNA polymerase.
Active in both forward or backward
orientation. Transcription factors are
proteins that bind specifically to
regulatory Sequences located in the promoter
. EX. SP1 for GC box AP1 for TGACTCA
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TRANSCRIPTION FACTORS FAMILIES Zinc-finger
domains they consist of repeats of cysteine and
histidine residues
that fold into looped structures (fingers)
that bind DNA.
Contain a zinc ion.
Found in SP1, Steroid Hormone
Receptors Helix-turn-helix one helix binds DNA,
while others stabilize the binding.
EX. Homeodomain proteins
(embryonic development) Leucine-zipper and
helix-loop-helix proteins contain dimerized
polypetide chains. Different members of these
two families can dimerize together.
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EUKARYOTIC REPRESSORS Bind to DNA (or protein)
and inhibit transcription. -Block the binding of
activators to regulatory sequences (by
competing) -Bind to transcription factors and
inhibit their DNA binding.
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Processing A newly made mRNA copy of a gene
(pre-mRNA), must undergo three major
modifications before it can leave the nucleus and
be translated into a protein1) Capping - a
special nucleotide is attached at the 5' end of
the mRNA. It is believed that this modification
is necessary for efficient initiation of protein
synthesis and serves as stabilization.2)
Poly(A)-tail - a special enzyme attaches a chain
of 150-200 adenine nucleotides to the 3' end of
the pre-mRNA directly after transcription. This
is primarily believed to increase the stability
and therefore prolong the lifetime of an mRNA
molecule. 3) Splicing - The removal of non
coding sequences, introns, from the pre-mRNA to
create mRNA, containing only the coding sequences
for a protein. This process is carried out by the
spliceosome. The splicosome is a special complex
made up by proteins and a catalytic kind of small
nuclear RNA molecules, the snRNAs.
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CAPPING
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3'-end Cleavage and Polyadenylation.Virtually
all mRNAs have a number of adenylate residues at
their 3'-end the poly(A)-tail. These A's are not
encoded in the genes. Instead, the pre-mRNA is
cleaved at a defined site and a poly(A)
polymerase adds 150-200 adenylate residues. The
most important function of the poly(A)-tail is
during translation. The length of the
poly(A)-tail can be regulated in the cytoplasm.
In some species, for example, the egg cell
stores mRNA in the cytoplasm for later use after
fertilization. The stored mRNA has a short
poly(A)-tail. Activation of the mRNA for
translation includes lengthening of the
poly(A)-tail. It has also been suggested that the
poly(A)-tail is involved in determining how long
an mRNA is present in the cell before it is
degraded.
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3'-cleavage and polyadenylation are closely
coupled to the termination of transcription.
Specific sequences in the pre-mRNA direct the
binding of protein factors to the pre-mRNA. A
conserved AAUAAA sequence is absolutely necessary
and there are also less well-conserved sequences
on both sides of the AAUAAA sequence, e.g. a
U-rich sequence downstream, called DSE
(Downstream Element). Some of these factors are
believed to bind to RNA polymerase II and travel
along during transcription.
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A special feature of the polyadenylation reaction
is that the poly(A) polymerase sticks poorly to
the end of the pre-mRNA until approximately 20
A's have been added. Then, poly(A)-binding
protein (PAB) has bound to the short poly(A)-tail
and the poly(A) polymerase is more firmly bound
until 150-200 A's are rapidly added. At this
stage the poly(A) polymerase again tends to fall
off.
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Summary CAPPING 5 end of the pre-mRNA
7-CH3-Guanosine
CBP20 and CBP80
CBC
Important for initiation of TRANSLATION 3end
cleavage and polyadenylation Important for
termination of transcription. Determine when the
RNA polymerase falls off the DNA
template. AAUAAA polyadenyl signal DSE
Downstream elements, less conserved, bind to RNA
pol.
PAB poly(A) binding protein
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SPLICING Introns (non coding sequences) are cut
out from the pre-mRNA.
http//nobelprize.org/educational_games/medicine/
dna/a/splicing/index.html
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RNA EDITING
Processing events, other than splicing, described
in mitochondrial mRNAs, chloroplasts mRNA of
higher plants, and nuclear mRNAs of some
mammalian genes. Editing involves single base
changes. Best known example apolipoprotein
B(lipid transport in the blood). Two forms exist
a longer, unedited form, transcribed in the
liver, and a smaller version (50 long) in the
intestine as a result of a change from a C to a U
that leads to a UAA (termination codon) and a
premature stop of protein synthesis.
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• RNA DEGRADATION
• While tRNA and rRNA are very stable, mRNAs are
  degraded at
• different rates, providing an additional
  parameter to the regulation
• of eukaryotic gene expression.
• Shortening of poly-A tail.
• Removal of the 5CAP
• Degradation of the mRNA from both ends.
• Half-life in mammalian is 30 min-20 hours.

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Ferritin Receptor Ferritin receptor is a cell
surface protein involved in the uptake of iron
(Fe). The level of ferritin receptor mRNA is
regulated by the availability of iron. If the
supply is adequate, the mRNA is rapidly degraded
by a nuclease at the 3 end. If iron is scarce, a
regulatory protein, called IRE-BP, binds to a
sequence near the 3 end of the mRNA, protecting
the mRNA from cleavage by the nuclease.
IRE-BP Iron Responsive Element-Binding Protein
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Regulation of ferritin receptor mRNA degradation