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 specificityNuclear locationSusceptibility to -amanitin
-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 threeribosomal 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 TATAboxes or a downstream promoter element (DPE)
Enhancer elements can be very distant fromthe start site
RNP I promoters transcribe ribosomal genesarranged 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 about15 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’ sideof 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 initiatorelement (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 distancesupstream and can function when presenton the antisense strand , in contrast to the -35 sequence in prokaryotes
Prokaryotic -10 and -35 bind RNP; eukaryoticCAAT 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 TFIIDbinding to TATA-box promoters
Binding of TBP induces conformational change in DNA to promote unwinding
Additional TFs bind TBP to form the basaltranscription 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 regulatetranscription
Multiple transcription factors interact witheukaryotic 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-boundactivators 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 havemany 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 femalesecondary sex characteristics and contribute to control of the ovarian cycle
Estrogens are relatively hydrophobic and candiffuse 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 domaincontaining 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 coactivatorbinding 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 targetsEstradiol is an “agonist”Anabolic steroids bind the androgen receptor to
stimulate development of lean muscleAntagonists bind nuclear hormone receptors to
act as competitive inhibitors of agonistsTamoxifen and raloxifene inhibit activation of the
estrogen receptor, used in treatment of breast cancer (selective estrogen receptormodulators - 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 looseningchromatin structure
Acetylated histone residues also interact with the “bromodomain”, a specific acetyllysine binding domain present in many eukaryotic transcription regulators
Bromodomains serve as docking sites to recruit proteins that affect transcription
Proteins that bind TBP are called TAFs (TATA-box- binding 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