Gene Expression

Levels of Regulation

• Gene expression is regulated by several different mechanisms.

• transcriptional control is (probably) the most important: binding of proteins to control regions adjacent to genes that cause RNA polymerase to transcribe the genes or not.

• post-transcriptional regulation includes control of RNA: splicing, transport to specific parts of the cell, stability; and protein: translation, processing, stability.

• long range controls: chromatin structure• epigenetic mechanisms: control that is inherited during

cell division but which doesn’t involve altering the DNA base sequence.

Transcriptional Control• The basic situation is that proteins binding

to DNA sequences near the gene cause or prevent transcription.

• Proteins that bind to DNA regulatory sequences and affect transcription are called transcription factors.

• Transcription factors, which migrate from the ribosomes to their site of action are said to act in trans. In contrast, the DNA regulatory sequences adjacent to the gene are said to act in cis, meaning that they only affect the gene they are attached to (and not other copies of the gene in the cell).

• Some transcription factors are general: involved in all transcription complexes, while others are specific: only used in certain tissues or with certain stimuli. The latter are often called “tissue-specific transcription factors”.

Cis-Acting DNA Sequences• The most important DNA regulatory

sequence is the promoter, the place where RNA polymerase binds and starts transcription.

• Five short sequences are conserved in eukaryotic promoters, but not all are found with all genes. All are close to the transcription start point, with some upstream and some downstream of it.

• The best known is the TATA box, located about 25 bp upstream from the transcription initiation point. Like all these elements, the TATA box is a consensus sequence, and it is not present in all genes.

• Tissue-specific sequences are usually upstream from the promoter, and consist of short consensus sequences.

Enhancers and Silencers

• Enhancers and silencers are tissue-specific cis-acting DNA sequences that increase or decrease transcription regardless of their position (within limits) or orientation: they can be either 5’ or 3’ to the gene itself.

• Enhancers and silencers work by bending the DNA to help transcription factors bind to the promoter.

Transcription Factors• Transcription factors generally have two

functional sections (domains): a DNA-binding domain that attaches to the specific DNA sequence, and an activation domain that stimulates transcription. The activation domain works by allowing other transcription factors to create the transcription complex.

• The DNA-binding domains fall into several general types, and proteins that have one of these domains are usually assumed to be transcription factors.

• Leucine zipper motif. An alpha helix that has a leucine every 7 amino acids, so all the leucines are on the same side of the molecule. This allows the protein to form a dimer by hydrophobic interactions. This dimer grips the DNA double helix

More Transcription Factors• Zinc finger motif: binds a Zn2+ ion

between two cysteines and two histidines (C2H2 proteins) or between four cysteines (C4 proteins). Sometimes a zinc finger protein will have more than one zinc finger motif.

• Helix-turn-helix motif consists of two alpha-helices connected by a short region of other amino acids. The two helices bind the DNA major groove. This is a common motif in homeobox gene regulation.

• Helix-loop-helix motif, which is different from the HTH motif. HLH has a much longer connecting loop that allows more flexibility in the molecule.

Yeast Two-Hybrid System• The yeast two-hybrid system is a way to

detect interactions between proteins. It is often used to find proteins that interact with the protein you are studying.

• A transcription factor that regulates the GAL4 gene (involved in galactose utilization) was split into separate DNA binding and activation domains.

• The “bait” protein (the protein you are studying) is fused to the binding domain.

• A large number of other protein-coding genes are fused to activation domains: a library of “prey” sequences.

• Each individual prey sequence is co-transformed into yeast along with the bait.

• If the bait and prey proteins interact in the cell, the attached DNA binding and activation domains will be brought together at the GAL4 gene, causing it to be transcribed. This event can be detected using a chromogenic (color-generating) substrate.

Signalling• How do events from the external world

affect gene transcription? Since the ability to respond to the external world is one of the primary hallmarks of life, it is not surprising that many different mechanisms exist. We will look at two basic mechanisms.

• Steroid response. – Steroids are small hydrophobic molecules

that diffuse readily through the cell membrane.

– Steroid receptors are found in the cytosol. – When the correct steroid hormone binds

to its receptor protein, the protein undergoes a conformational shift and migrates to the nucleus.

– There it binds to cis-acting hormone response elements (HRE) and stimulates transcription.

– Steroid receptors have 3 domains: ligand (steroid) binding, DNA-binding, and activation.

– Retinoic acid (vitamin A and derivatives) and thyroxin (thyroid hormone) use the same basic system.

Signal Transduction• Peptide hormones are hydrophilic

and don’t diffuse through the membrane. Instead, they bind to receptor proteins on the cell surface.

• The receptor proteins then induce a cascade of protein interactions and modifications that eventually reaches transcription factors in the nucleus.

• Most of the interactions involve the phosphorylation of proteins by kinases (which activates the proteins) or de-phosphorylation by phosphatases (which deactivates them).

• “Second messengers”, small molecules that transmit a signal within the cell are often involved. The best known second messengers are cyclic AMP (cAMP) and phospholipids.

G Protein Signaling• One major class of signal transduction

pathway involves G proteins. • The G protein receptor is a “serpentine”

protein that passes through the membrane 7 times. The ligand binding portion in on the outside, and the effector region is on the inside of the cell.

• In addition to working with peptide hormones, serpentine receptors also are used for the olfactory system, which is the largest gene family in humans: over 900 genes.

• When the ligand binds to the receptor, it changes conformation and interacts with the G protein.

• The G protein is usually in an inactive state. • It is activated by an interaction with the

receptor, and it spontaneously reverts to inactive state after a very short time. The G protein is thus thought to be the critical link that rapidly responds to changes in environmental conditions.

More G Protein Signalling• A G protein is a trimer of alpha, beta, and

gamma subunits, bound to the cytoplasmic face of the membrane by covalently attached fatty acids.

• Alpha binds GDP in the normal, unactivated state.

• When the receptor interacts with the G protein, it causes the G protein alpha subunit to release the GDP and bind a GTP (the concentration of GTP is much higher than that of GDP in the cell).

• Binding the GTP causes the G protein trimer to dissociate into two parts: alpha+GTP, and beta-gamma.

• Alpha slowly hydrolyzes the GTP back to GDP, which causes the trimer to re-form.

• While the subunits are separate they are active.

• In some cases, alpha interacts with the next protein in the signaling pathway, and in other cases beta-gamma interacts with the next protein.

More G Protein Signaling• In the case of adenylate cyclase,

activation is accomplished by the alpha+GTP subunit. Adenylate cyclase then creates cAMP, an intracellular second messenger that activates certian kinases.

• In other cases, the G protein alpha subunit activates phospholipase C, which cleaves phosphatidyl inositol bisphosphate (PIP2), a phospholipid into diacyl glycerol and inositol triphosphate (IP3).

– Diacyl glycerol mobilizes Ca2+ ions from the endoplasmic reticulum, by opening a calcium ion channel. Calcium bound to calmodulin in turn activates a kinase cascade.

– IP3 activates a protein kinase which in turn activates other kinases in a cascade that ends up activating transcription factors in the nucleus.

• Cell signaling is a large area: we have only touched on a small part of it.

RNA Regulation• At least half of all human genes are

expressed in different ways in different tissues. Different transcriptional start sites, different intron splicing patterns, and different poly A addition sites can give quite a few different proteins from the same gene.

• Different proteins from the same gene are called isoforms.

• Isoforms are produced in different tissues, different times in development, different subcellular locations (soluble vs. membrane-bound, for instance), etc.

• Dystrophin, the Duchenne muscular dystrophy protein, has at least 7 different transcription start sites, used in different tissues. (B, brain; M, muscle; P, Purkinje; R, retina; B,K, brain and kidney; S, Schwann cells; G, general)

• A good example of alternate splicing patterns in different tissues is tropomyosin, which has 5 optional exons. Tropomyosin is a protein in striated muscle that binds to actin and prevents it from interacting with myosin: thus it regulates muscle movements.

Control of Alternative Splicing

• RNA splicing is performed by snRNPs, small nuclear ribonucleoprotein complexes, which are RNA/protein hybrids.

• Variations in snRNPs occur in different cells and recognize slightly different splicing signals.

• Some of the splicing proteins also assist in transporting mRNA out of the nucleus.

Messenger RNA Stability• micro RNAs are a major cause of

messenger RNA decay in the cell. • miRNAs are produced from RNA-

only genes. The RNA forms a stem-loop structure.

• the Dicer enzyme processes the double-stranded region, incorporating one strand of the RNA into the RISC complex.

• The miRNA in the RISC complex is complementary to (antisense) the 3’ region of a specific messenger RNA.

• The RISC complex binds to the messenger RNA and degrades it.

• Alternatively, the RISC complex can inhibit translation of the messenger RNA

Translational Control• Regulation of whether the messenger

RNA is translated or not.• The best studied example is ferritin, a

protein that stores up to 4500 iron atoms (as iron hydroxyphosphate) in its center.

• The ferritin mRNA contains an iron-response element in the 5’ UTR. The IRE folds up into a hairpin loop, which can bind to the IRE-binding protein. When iron levels are low, IRE-BP binds and prevents translation of the mRNA. This allows the ferritin mRNA to remain intact while preventing any further sequestration of iron atoms.

• Transferrin is the major iron-carrying protein in the blood serum.

• The transferin mRNA contains 3 IREs in the 3’ UTR. RNA degradation is prevented by IRE-BP binding.

Control of Protein Degradation• To react quickly to the environment, a cell

must be able to remove outdated signals quickly. Many proteins, especially regulatory signaling proteins, are degraded by ubiquitin-mediated proteolysis.

• Ubiquitin is a small protein that is highly conserved in evolution.

• In this system, multiple copies of ubiquitin are covalently attached to the target protein in long chains. The complex is then transported to the proteosome, a large multi-subunit barrel-shaped structure. The proteosome degrades the target protein to amino acids and recycles the ubiquitin.

• Target specificity is provided by the enzyme that attaches ubiquitin to the target proteins: there are hundreds of different E3-ubiquitin ligases.

– One target is hydrophobic amino acids that are normally buried in the protein’s interior or within membranes.

– N-end rule: On average, a protein's half-life correlates with its N-terminal residue.

• Proteins with N-terminal Met, Ser, Ala, Thr, Val, or Gly have half lives greater than 20 hours.

• Proteins with N-terminal Phe, Leu, Asp, Lys, or Arg have half lives of 3 min or less.

The proteosome also re-folds misfolded proteins if the proteins are protected from degradation by chaperone proteins. Misfolding is a common result of heat shock.Ubiquitin plays a number of other roles in the cell, including cell signaling and X chromosome inactivation.

Chromatin Effects

• Recall that chromosomal DNA is wrapped up in nucleosomes: 8 histone proteins with about 150 bp of DNA wrapped around them. Higher level packaging also exists. All of this structure makes it difficult for RNA polymerase and transcription factors to reach the target DNA.

• We will look at several mechanisms that affect chromatin structure: chromatin remodeling, histone modification, and DNA methylation.

Chromatin Remodeling• Moving nucleosomes around to

allow transcription factors to reach the cis acting regulatory sites is accomplished by large protein structures called chromatin remodeling complexes.

• Remodeling slides nucleosomes along the DNA, away from the region of the promoter. The process uses ATP.

• The DNA exposed by moving histones away is more accessible for restriction enzymes and DNase in teh lab: “DNase hypersensitive sites” are a sign of active genes.

Histone Acetylation• Histones are basic proteins: lysines have a

+ charge that is attracted to the – charges on DNA phosphates.

• Histone acetylases add acetate (CH3COOH) to the NH2 at the end of lysine. This removes the + charge, and in consequence the histones are less tightly bound to the DNA.

• Genes in the region of acetylated histones are active; non-acetylated histones are associated with inactive genes. The chromatin in areas of acetylated histones is less condensed.

• Histone acetylases and de-acetylases can be part of transcriptional complexes, helping to activate specific genes.

• Histones are also subject to methylation and phosphorylation, both of which affect gene activity. There are several different sites of methylation on histones, and which ones are methylated or not might be important for specific regulatory events.

DNA Methylation• DNA methylation is the addition of methyl groups to

cytosine, creating 5-methyl cytosine. In mammalian DNA this almost always occurs when the C is followed by a G: CpG.

• The methylation state of DNA is maintained through mitosis: daughter cells are methylated in the same way as the parent cell. Methylation changes are thus epigenetic changes: heritable changes that don’t alter the DNA base sequence.

• When DNA replicates, an enzyme called “maintainence methylase” recognizes methylated cytosines on the old strand (in a CpG dinucleotide), and methylates the corresponding C on the new strand.

• In bacteria, DNA methylation is used as a defense against foreign DNA: the cell’s DNA is methylated at specific sites, and any non-mehtylated DNA is cut by restriction enzymes that recognize the same sites.

• DNA methylation is associated with inactive genes

• DNA from sperm and egg are heavily methylated, but all methylation is removed in the early embryo (morula and early blastocyst). Shortly thereafter new methylation patterns are imposed on different cell lineages. These patterns pernanently inactivate some of the genes.

Methylation and Imprinting• Imprinting is a situation where the maternal

and paternal alleles are not expressed identically in the embryo. Imprinting is caused by different methylation patterns in the DNA of the egg and sperm.

• This seems to be the major reason why uniparental diploid embryos do not produce viable offspring: some genes require an active, unmethylated gene from the father while others need an active gene from the mother.

• Prader-Willi syndrome and Angelman syndrome are both caused by deletions or uniparental disomy of 15q. Prader-Willi results when only the maternal gene is active, and Angelman when only the paternal is active.

– PWS is characterized by obesity due to an insatiable appetite, small hands and feet, short stature, and hypogonadism. In addition, there is a common behavioral phenotype, including temper tantrums, stubbornness, and controlling and manipulative behavior.

– AS is characterized by severe mental retardation, severe speech impairment, and unsteady gait and/or tremulousness of the limbs. In addition, individuals with AS present with inappropriate laughter and excitability.