Download File

10-31-2011 Gene expression in eukaryotes
1. Eukaryotic RNA polymerases
2. Regulation of eukaryotic RNP
3. Hormonal regulation
4. Histone acetylation
Special features of eukaryotic gene
expression
1. Complex transcriptional control
2. RNA processing
3. The nuclear membrane creates opportunities
for temporal and spatial regulation
Eukaryotic RNA polymerases
Three eukaryotic RNPs differ in:
Template specificity
Nuclear location
Susceptibility to a-amanitin
a-amanitin binds strongly to RNP II, inhibits
elongation phase of RNA synthesis
(mRNAs, snRNAs)
Eukaryotic RNPs are large, containing 8-14
subunits
RNP II is nucleoplasmic, synthesizes mRNA
and several small nuclear snRNAs
RNP I is located in nucleoli, transcribes three
ribosomal rRNAs (18S, 23S and 5.8S) as
a single transcript
RNP III is nucleoplasmic, synthesizes
ribosomal 5S rRNA and transfer tRNAs
RNP II contains a unique C-terminal domain
The CTD contains multiple repeats of the
consensus sequence YSPTSPS
The activity of RNP II is regulated by
phosphorylation of serine residues in the
CTD
Phosphorylation of the CTD enhances
transcription and recruits factors needed
to process RNP II products
Eukaryotic genes contain promoters
Eukaryotic promoters attract RNPs to start
sites
Promoters are cis-acting elements (on the
sameDNA molecule as the gene)
Eukaryotic promoters differ in structure and
provided the basis for the template
specificity of the three different RNPs
RNP II promoters have conserved sequence
elements that define the start site:
Initiator elements (Inr) are assisted by TATA
boxes or a downstream promoter
element (DPE)
Enhancer elements can be very distant from
the start site
RNP I promoters transcribe ribosomal genes
arranged in multiple tandem repeats,
each containing a copy of the three rRNA
genes
Promoters are located in stretches of DNA that
separate the rRNA genes repeats
The transcriptional start site is marked by the
ribosomal initiator element (rInr)
An upstream promoter element (UPE) joins
with the rInr to bind proteins that recruit
RNP I
RNP III promoters are located within the
transcribed gene sequence, downstream
of the start site
Type I promoters are found in the 5S rRNA
gene and contain two short sequences,
the A block and the C block
Type II promoters are found in tRNA genes
and consist of two 11-bp sequences, the
A block and the B block, located about
15 bp from either end of the gene
RNA polymerase II requires complex regulation
Regulation of RNP II accounts for cellular
differentiation and specific gene expression
RNP II promoters are located on the 5’ side
of the start site
The TATA box lies between positions -30 and
-100 upstream from the start site
The TATA box is often paired with an initiator
element (Inr) near the start site
A downstream core promoter element (DPE) is
present between positions +28 to +32 when
TATA is absent
RNP II is regulated by additional upstream
elements between -40 and -150
Many RNP II promoters contain a CAAT box
and some contain a GC box
Constitutive genes tend to have GC boxes
CAAT and GC boxes lie at variable distances
upstream and can function when present
on the antisense strand, in contrast to
the -35 sequence in prokaryotes
Prokaryotic -10 and -35 bind RNP; eukaryotic
CAAT and GC boxes bind protein factors
The TFIID protein complex initiates assembly
of an active transcription complex
Transcription factors bind cis-acting elements
to help regulate eukaryotic genes
TATA-box-binding protein (TBP) initiates TFIID
binding to TATA-box promoters
Binding of TBP induces conformational
change in DNA to promote unwinding
Additional TFs bind TBP to form the basal
transcription apparatus
Phosphorylation of RNP II CTD begins elongation
TBP bound to DNA
Enhancers stimulate transcription thousands
of bases away from the start site
Enhancers greatly increase promoter activity
Enhancers may be located upstream,
downstream or within transcribed genes
Enhancers may be on either DNA strand
Enhancers are bound by proteins that regulate
transcription
Multiple transcription factors interact with
eukaryotic promoters and enhancers
High transcription rates are attained by binding of
transcription factors to specific genes
Transcription factors are often expressed in a
tissue-specific manner
Eukaryotic TFs function by recruiting other proteins
to build large complexes that interact with the
transcriptional machinery to activate or
repress transcription
Mediators act as a bridge between enhancer-bound
activators and promoter-bound RNP II
“Combinatorial control” is attained when
multiple independently regulated TFs
function cooperatively to regulate
transcription
A specific TF can have different effects
depending on other TFs expressed the cell
Important for multicellular organisms that have
many different cell types
Humans have only 33% more genes that the
worm C. elegans, demonstrating that
regulation rather than gene content
governs cellular diversity
Gene expression is regulated by hormones
Eukaryotic cells respond to external stimuli to
regulate genes
Initiation of transcription by RNP II is responsive
to many signal transduction pathways
(eg, STAT5 via tyrosine kinase activation)
Estrogens control the development of female
secondary sex characteristics and
contribute to control of the ovarian cycle
Estrogens are relatively hydrophobic and can
diffuse through cell membranes
Inside cells estrogens bind to estrogen receptors
Estrogen receptors are soluble and located in the
cytoplasm or nucleoplasm
Estrogen receptors are part of a large family that
includes testosterone, thyroid hormones and
retinoids
On binding the signal molecule (ligand) the
receptor-ligand complex binds to control
elements in DNA to modify the expression of
specific genes
Humans make 50 such “nuclear hormone receptors”
Nuclear hormone receptors have similar
domain structures
Nuclear hormone receptors bind specific sites
in DNA called “response elements”
Estrogen response elements contains the
consensus sequence:
5’-AGGTCANNNTGACCT-3’
Estrogen receptors have a ligand binding
domain and a DNA binding domain
containing zinc fingers
Binding of estradiol to the ligand binding domain induces
a conformational change that allows the receptor to
recruit other proteins that stimulate transcription
Nuclear hormone receptors recruit
coactivators and corepressors
Coactivators bind to the receptor only after it
has bound ligand to form a coactivator
binding site
Receptors for thyroid hormone and retinoic
acid repress transcription when not
bound to hormone
Repression is mediated by the ligand binding
domain
In the unbound form the ligand binding domain
binds to corepressor proteins that
inactivate transcription
Binding of ligand triggers release of the
corepressor freeing the ligand binding
domain to bind coactivators
Steroid hormone receptors are drug targets
Estradiol is an “agonist”
Anabolic steroids bind the androgen receptor to
stimulate development of lean muscle
Antagonists bind nuclear hormone receptors to
act as competitive inhibitors of agonists
Tamoxifen and raloxifene inhibit activation of the
estrogen receptor, used in treatment of
breast cancer (selective estrogen receptor
modulators - SERMs)
Histone acetylation results in chromatin
remodeling
Histone acetyltransferases (HATs) attach acetyl
groups to lysine residues in histones
Histone acetylation neutralizes the ammonium
group on the histone to an amide group,
reducing affinity for DNA and loosening
chromatin structure
Acetylated histone residues also interact with the
“bromodomain”, a specific acetyllysine
binding domain present in manyeukaryotic
transcription regulators
Bromodomains serve as docking sites to recruit
proteins that affect transcription
Proteins that bind TBP are called TAFs (TATA-boxbinding protein associated factors)
TAF1 contains two bromodomains that bind
acetylated lysine residues in histone H4
Acetylated histone residues also bind to
bromodomains in chromatin remodeling
machines
Chromatin remodeling machines are ATPases
that use the energy of ATP hydrolysis to
move nucleosomes along DNA, exposing
binding sites for other factors
Histone acetyltransferases activate
transcription in three ways:
1. Reducing affinity of histones for DNA
2. Recruiting other components of the
transcriptional machinery
3. Initiating the remodeling of chromatin
Histone deacetylases contribute to transcriptional
repression by reversing the effects of HATs