Transcript Transcription Initiation

Psalm 102:24
25 In the beginning you
laid the foundations of
the earth, and the
heavens are the work of
your hands.
©2001 Timothy G. Standish
Initiation of
Transcription:
In Eukaryotes
Timothy G. Standish, Ph. D.
©2001 Timothy G. Standish
Expression Control In Eukaryotes
Some of the general methods used to control
expression in prokaryotes are used in
eukaryotes, but nothing resembling operons is
known
Eukaryotic genes are controlled individually
and each gene has specific control sequences
preceding the transcription start site
In addition to controlling transcription, there
are additional ways in which expression can
be controlled in eukaryotes
©2001 Timothy G. Standish
Eukaryotes Have Large
Complex Geneomes
The human genome is about 3 x 109 base
pairs or ≈ 1 m of DNA
Because humans are diploid, each nucleus
contains 6 x 109 base pairs or ≈ 2 m of DNA
Some gene families are located close to one
another on the same chromosome
Genes with related functions appear to be
distributed almost at random throughout the
the genome
©2001 Timothy G. Standish
Highly Packaged DNA Cannot
be Expressed
Because of its size, eukaryotic DNA
must be packaged
Heterochromatin, the most highly
packaged form of DNA, cannot be
transcribed, therefore expression of
genes is prevented
Chromosome puffs on some insect
chomosomes illustrate areas of active
gene expression
©2001 Timothy G. Standish
Only a Subset of Genes is
Expressed at any Given Time
It takes lots of energy to express genes
Thus it would be wasteful to express all
genes all the time
By differential expression of genes, cells
can respond to changes in the environment
Differential expression, allows cells to
specialize in multicelled organisms.
Differential expression also allows
organisms to develop over time.
©2001 Timothy G. Standish
Control of Gene Expression
Cytoplasm
Packaging
Degradation
DNA
Transcription
Transportation
Modification
RNA
RNA
Processing
mRNA G
G
AAAAAA
Nucleus
Export
Degradation etc.
AAAAAA
Translation
©2001 Timothy G. Standish
Logical Expression Control Points
Increasing cost
DNA packaging
Transcription
RNA processing
mRNA Export
mRNA masking/unmasking and/or
modification
mRNA degradation
Translation
Protein modification
Protein transport
Protein degradation
The logical
place to
control
expression is
before the
gene is
transcribed
©2001 Timothy G. Standish
Three Eukaryotic
RNA Polymerases
1 RNA Polymerase I - Produces rRNA in
the nucleolus, accounts for 50 - 70 % of
transcription
2 RNA Polymerase II - Produces mRNA
in the nucleoplasm - 20 - 40 % of
transcription
3 RNA Polymerase III - Produces tRNA
in the nucleoplasm - 10 % of
transcription
©2001 Timothy G. Standish
A “Simple” Eukaryotic Gene
Transcription
Start Site
3’ Untranslated Region
5’ Untranslated Region
Introns
5’
Exon 1 Int. 1
Promoter/
Control Region
Exon 2
3’
Int. 2 Exon 3
Exons
Terminator
Sequence
RNA Transcript
©2001 Timothy G. Standish
Enhancers
DNA
Many bases
5’
3’
Enhancer
5’
Promoter
TF
Transcribed Region
3’
TF
5’
TF TF RNA
RNA
Pol.
Pol.
5’
3’
RNA
©2001 Timothy G. Standish
Eukaryotic RNA Polymerase II
RNA polymerase is a very fancy enzyme that
does many tasks in conjunction with other
proteins
RNA polymerase II is a protein complex of
over 500 kD with more than 10 subunits:
©2001 Timothy G. Standish
Eukaryotic RNA Polymerase II
Promoters
Several sequence elements spread over about
200 bp upstream from the transcription start
site make up RNA Pol II promoters
Enhancers, in addition to promoters,
influence the expression of genes
Eukaryotic expression control involves many
more factors than control in prokaryotes
This allows much finer control of gene
expression
©2001 Timothy G. Standish
Initiation
T. F.
Promoter
T. F.
RNA
Pol. II
RNA
Pol. II
mRNA
5’
©2001 Timothy G. Standish
Eukaryotic Promoters
Promoter
5’
Exon 1
Sequence elements
TATA
~200 bp
“TATA Box”
Initiator
Transcription
start site
SSTATAAAASSSSSNNNNNNNNNNNNNNNNNYYCAYYYYYNN
(Template strand)
~-25
-1+1
S = C or G
Y = C or T
N = A, T, G or C
©2001 Timothy G. Standish
Initiation
TFIID Binding
TFIID
“TATA Box”
Transcription
start site
TBP Associated
Factors (TAFs)
-1+1
TATA Binding
Protein (TBP)
©2001 Timothy G. Standish
Initiation
TFIID Binding
Transcription
start site
TFIID
-1+1
80o Bend
©2001 Timothy G. Standish
Initiation
TFIIA and B Binding
TFIID
TFIIB
Transcription
start site
-1+1
TFIIA
©2001 Timothy G. Standish
Initiation
TFIIF and RNA Polymerase Binding
TFIID
TFIIB
Transcription
start site
-1+1
TFIIA
TFIIF
RNA Polymerase
©2001 Timothy G. Standish
Initiation
TFIIE Binding
TFIIF TFIIB
RNA Polymerase
-1+1
TFIIA
TFIIE
TFIID
Transcription
start site
TFIIE has some
helicase activity and
may by involved in
unwinding DNA so
that transcription can
start
©2001 Timothy G. Standish
Initiation
TFIIH and TFIIJ Binding
TFIIJ
TFIIH
TFIIF TFIIB
P
TFIIA
PP
RNA Polymerase
-1+1
TFIIE
TFIID
Transcription
start site
TFIIH has some
helicase activity and
may by involved in
unwinding DNA so
that transcription can
start
©2001 Timothy G. Standish
Initiation
TFIIH and TFIIJ Binding
TFIIJ
TFIIH
TFIIF TFIIB
P
PP
-1+1
TFIIE
TFIID
Transcription
start site
RNA Polymerase
TFIIA
©2001 Timothy G. Standish
Initiation
TFIIH and TFIIJ Binding
Transcription
start site
P
-1+1
PP
RNA Polymerase
©2001 Timothy G. Standish
©2001 Timothy G. Standish