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  -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 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-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