Transcript transcription factors

Gene Expression
1: Activation (overview),
Transcription, Translation.

The Goodwin Equations
Review
 An active gene is one that is being
transcribed, and whose transcription product
is being translated, and whose translation
product affects cellular behavior/development.
 Genes are activated by transcription factors
– the principal way transcription is controlled
 Transcription factors are proteins that
themselves are gene products.
 Transcription factors often require a cofactor,
a very important environmental signal.
Gene Activation is a control system
Transcription factor  Active Gene 
Transcription Product:
 Messenger RNA
 


Translation Product:
 (Active Protein)  


Change in

 Change in 
 transcription factor 

cofactor level 




activity

Thus, one has a rudimentary feedback loop
Since the transcription factor is itself a
gene product the feedback loop can
(and usually does) involve multiple
genes.
Since the cofactor level may involve
environmental stimuli the feedback
loop can involve their signal
transduction pathways.
Gene Expression is best
understood by mapping,
quantifying, and understandings
the behavior of these loops.
Transcription
A good reference:
(Blackwell, 2001)
Genes are transcribed (copied) into
'messenger RNA' by an enzyme called
RNA Polymerase [RNAP].
Polymerase binds to a 'promoter region' at
the beginning of a gene.
The polymerase traverses the gene,
copying as it goes.
Polymerase normally leaves DNA at the
gene's end.
Multiple polymerases may be attached to
one gene at any given time.
The
transcription
“bubble”,
White, Fig.
1.8
Three kinds of RNAP
Pol I – rRNA
Pol II – mRNA and snRNA (small nuclear
RNA, involved in splicing)
Pol III – small RNA’s (tRNA, 5s rRNA …)
We will concentrate on Pol II although many
features are common to all three.
Model of Pol II RNAP (White)
What determines the rate of
transcription?
 Transcription velocity is mostly constant, over
one gene and from gene to gene.
 Transcription length is determined by the
gene. Thus …
 (Molar) synthesis rate for transcription is
controlled by gene length, number of RNAP's on
the gene.
Rates (Hargrove): 2500 nt min-1 (procaryotes),
3000 nt min -1 (eukaryotes)
The initiation rate for transcription*
is the most important factor
determining which gene products
are generated
* i.e. the attachment and hence (in steady state)
the detachment rate for RNA polymerase
(RNAP). This determines the average number
of mRNA's on the gene.
What determines the rate of
translation (mRNA protein)?
 Translation is effected by ribosomes,
complex enzymes made of both protein
and nucleic acid, that traverse mRNA's
and translate their codons (RNA triplets)
into amino acid sequences.
 In a manner similar to transcription, the
ribosome traverses at a near-constant
velocity. Thus …
for translation
…
(Molar) synthesis rate is controlled by
ribosomal attachment rate, mRNA length, and
the number of mRNA's present.
Rates (Hargrove): 2700 nt min-1
(procaryotes), 720 nt min -1 (eukaryotes)
Summary of Gene Expression Rates
Typical values for parameters and rates needed to
quantify gene expression in E. coli and mammalian
cells (from Hargrove)
(nt = nucleotides)
E. coli
Mammals
Initiation
1 sec-1
10 min -1
Transcription
2500 nt min-1
3,000 nt min-1
RNA processing
not applicable
ca. 10 min
Half-life, nuclear RNA not applicable
ca. 10 min
Nucleocytoplasmic
transport
not applicable
ca. 10 min
Half-life, mRNA
1-3 min
1-20 h
mRNA translation
2700 nt min-1
720 nt min-1
Half-life, protein
20-60 min
2-100 h
The train-on-the-track model
GENE (DNA)
m
R
N
A
RNA polymerase
Pr o
tein
Pro
du c
t
Ribosome
Rate = Number of tracks x Number of trains x Velocity of trains / Track length
Critical Factors:
For transcription – the attachment rate,
since the number of gene copies (one or
two), transcription velocity and length are
fixed.
For translation – the number of mRNA's
present, since the ribosomal attachement
rate, translation velocity and length are
fixed.
Summary of Steps in Eukaryotic Processing
What is the RNAP “train starter”?
 Transcription factors.
Inducers
Repressors
These are protein molecules, made by genes,
that bind to a gene at an operator site, in or near
a promoter region, upstream of where transcription takes place. They often exist in two forms
quiescent and active. Usually a small molecule
induces the change:
Inactive factor  small molecule  active factor
Transcription Factors
It is important to remember that transcription factors are
proteins, come from genes (like all proteins), and may influence
either their predecessor gene or –often– other genes.
Summary of the structure of
the Engrailed homeodomain
bound to DNA, as revealed by
X-ray crystallography.
Cylinders represent the three
-helices of the homeodomain,
ribbons represent the sugar
phosphate backbone of the
DNA and bars symbolize the
base pairs. The recognition
helix (3) is shown in red.
Transcription
factors have
many shapes
and thus modes
of interaction
with DNA
Promoters (pol II)
Contain multiple binding sites for
transcription factors.
Other binding sites upward, downward of
(enhancers), and within transcribed region.
Basal Transcription
Factors
 TATA box – TATAa/tAa/ t
 Initiator – YYANa/ tYY,
where Y is a pyrimidine, N
is any base, and
transcription begins at the
A
 Picture shows assembly
of basal pol II on
adenovirus ML promoter
Preassembly of the Complex is
possible: kinetic implications?
Actual Initiation (White, p. 62)
Promoter melting – DNA strands separate.
Initiation – first RNA phosphodiester bond is
formed.
Clearance – pol II released from the factors
assembled at the promoter.
Elongation and Termination
Elongation factors
Termination Sequence (AAUAAA) and cleavage
factor. Polyadenylation.
Enhancers (White, p. 73)
Enhancer region located ~ 1 kb upstream of mouse
muscle creatine kinase gene. There are at least 6
different transcription factors with expression
“governed by combinatorial interactions amongst the
transcription factors”.
Transcription factor activation
Transcription Factor Production,
one example. (White, p. 170)
Transcription
Control by
Stimulated
Translocation
Transcription of the WT1 Gene
Negative feedback: WT1 protein inhibits expression
of its own gene and also that of PAX-2 an activator of
th WT1 promoter.
Myogenesis
Upstream regulators force
differentiation to
mesodermal precursor
cells that then express
bHLH proteins that
stimulate transcription of
their own genes. They also
activate genes that make
MEF2, which further
accelerates transcription of
genes for bHLH proteins.
MEF2 and bHLH proteins
both stimulate other
muscle-specific genes.
Positive feedback!
In general, transcription factors and the
molecules that activate them are crucial to
determining the array of genes that are on.
Transcription factors
determine mRNA production;
do they determine mRNA
number?
(mRNA number is what
determines translation)
mRNA number is determined by a
balance between generation and
consumption rate:
d (mRNA)
 Production rate  k (mRNA)
dt
This is the first part of the Goodwin equation set.
mRNA numbers determine
protein production; do they
determine translation product
levels?
Translation product levels are
determined by a balance between
their generation and consumption
rates:
d (product)
 Production rate  k (product)
dt
First-order decay processes are almost always
assumed. The decay constant, however, may be
fixed by other situations within the cell.
This is the second part of the Goodwin equation set.
Frequently a translation
product will mediate the level
of another metabolite
(galactosidase, tryp enzymes)
 This can give rise to a third set of
Goodwin equations.
Product levels determined by the third set
often provide the "loop-closing" feedback.
What has been covered?
Transcription factor  Active Gene 
Transcription Product:
 Messenger RNA
 


Translation Product:
 (Active Protein)  


Change in

 Change in 
 transcription factor 

cofactor level 




activity
