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