Chapter 8- Control of Gene Expression Notes

An overview of gene expression- Gene Expression Can Be Regulated at Various Steps from DNA to RNA to Protein Example: Figure 8-3: Gene expression in eukaryotic cells can be controlled at various steps.

*Gene expression in eukaryotic cells can be controlled at various steps.* Examples of regulation at each of these steps are known, although for most genes the main site of control is step 1—transcription of a DNA sequence into RNA.

How transcriptional switches work- Transcriptional Switches Allow Cells to Respond to Changes in Their Environment Example: Figure 8-7: Genes can be switched off by repressor proteins.

*Genes can be switched off by repressor proteins.* If the concentration of tryptophan inside a bacterium is low (left), RNA polymerase (blue) binds to the promoter and transcribes the five genes of the tryptophan operon. However, if the concentration of tryptophan is high (right), the repressor protein (dark green) becomes active and binds to the operator (light green), where it blocks the binding of RNA polymerase to the promoter. Whenever the concentration of intracellular tryptophan drops, the repressor falls off the DNA, allowing the polymerase to again transcribe the operon. The promoter contains two key blocks of DNA sequence information, the -35 and -10 regions, highlighted in yellow, which are recognized by RNA polymerase (see Figure 7-10). The complete operon is shown in Figure 8-6.

How transcriptional switches work- Repressors Turn Genes Off and Activators Turn Them On Example: Figure 8-8: Genes can be switched on by activator proteins.

*Genes can be switched on by activator proteins.* An activator protein binds to a regulatory sequence on the DNA and then interacts with the RNA polymerase to help it initiate transcription. Without the activator, the promoter fails to initiate transcription efficiently. In bacteria, the binding of the activator to DNA is often controlled by the interaction of a metabolite or other small molecule (red triangle) with the activator protein. The Lac operon works in this manner, as we discuss shortly.

The molecular mechanisms that create specialized cell types- Epigenetic Mechanisms Allow Differentiated Cells to Maintain Their Identity Example: Figure 8-23: Histone modifications may be inherited by daughter chromosomes.

*Histone modifications may be inherited by daughter chromosomes.* When a chromosome is replicated, its resident histones are distributed more or less randomly to each of the two daughter DNA double helices. Thus, each daughter chromosome will inherit about half of its parent's collection of modified histones. The remaining stretches of DNA receive newly synthesized, not-yet-modified histones. If the enzymes responsible for each type of modification bind to the specific modification they create, they can catalyze the spread of this modification on the new histones. This cycle of modification and recognition can restore the parental histone modification pattern and, ultimately, allow the inheritance of the parental chromatin structure. This mechanism may apply to some but not all types of histone modifications.

How transcriptional switches work

-Transcription Regulators Bind to Regulatory DNA Sequences -Transcriptional Switches Allow Cells to Respond to Changes in Their Environment -Repressors Turn Genes Off and Activators Turn Them On -An Activator and a Repressor Control the Lac Operon -Eukaryotic Transcription Regulators Control Gene Expression from a Distance -Eukaryotic Transcription Regulators Help Initiate Transcription by Recruiting Chromatin-Modifying Proteins

Post-transcriptional controls- Each mRNA Controls Its Own Degradation and Translation Example: Figure 8-24: A bacterial gene's expression can be controlled by regulating translation of its mRNA.

*A bacterial gene's expression can be controlled by regulating translation of its mRNA.* (A) Sequence specific RNA-binding proteins can repress the translation of specific mRNAs by keeping the ribosome from binding to the ribosome-binding sequence (orange) in them RNA. Some ribosomal proteins exploit this mechanism to inhibit the translation of their own mRNA. In this way, "extra" ribosomal proteins those not incorporated into ribosomes—serve as a signal to halt their synthesis. (B) An mRNA from the pathogen Listeria monocytogenes contains a "thermosensor" RNA sequence that controls the translation of a set of mRNAs produced by virulence genes. At the warmer temperature that the bacterium encounters inside its human host, the thermosensor sequence denatures, exposing the ribosome-binding sequence, so the virulence proteins are made.

How transcriptional switches work- Transcriptional Switches Allow Cells to Respond to Changes in Their Environment Example: Figure 8-6 (Tryp operon): A cluster of bacterial genes can be transcribed from a single promoter.

*A cluster of bacterial genes can be transcribed from a single promoter.* Each of these five genes encodes a different enzyme; all of the enzymes are needed to synthesize the amino acid tryptophan. The genes are transcribed as a single mRNA molecule, a feature that allows their expression to be coordinated. Clusters of genes transcribed as a single mRNA molecule are common in bacteria. Each of these clusters is called an operon because its expression is controlled by a regulatory DNA sequence called the operator (green), situated within the promoter. The yellow blocks in the promoter represent DNA sequences that bind RNA polymerase.

The molecular mechanisms that create specialized cell types- Specialized Cell Types Can Be Experimentally Reprogrammed to Become Pluripotent Stem Cells Example: Figure 8-18: A combination of transcription regulators can induce a differentiated cell to de-differentiate into a pluripotent cell.

*A combination of transcription regulators can induce a differentiated cell to de-differentiate into a pluripotent cell.* The artificial expression of a set of four genes, each of which encodes a transcription regulator, can reprogram a fibroblast into a pluripotent cell with ES cell-like properties. Like ES cells, such iPS cells can proliferate indefinitely in culture and can be stimulated by appropriate extracellular signal molecules to differentiate into almost any cell type in the body.

An overview of gene expression Figure 8-1: A neuron and a liver cell share the same genome.

*A neuron and a liver cell share the same genome.* The long branches of this neuron from the retina enable it to receive electrical signals from many other neurons and carry them to many neighboring neurons. The liver cell, which is drawn to the same scale, is involved in many metabolic processes, including digestion and the detoxification of alcohol and other drugs. Both of these mammalian cells contain the same genome, but they express many different RNAs and proteins.

The molecular mechanisms that create specialized cell types- Epigenetic Mechanisms Allow Differentiated Cells to Maintain Their Identity Example: Figure 8-20: A positive feedback loop can create cell memory.

*A positive feedback loop can create cell memory.* Protein A is a master transcription regulator that activates the transcription of its own gene—as well as other cell-type-specific genes (not shown). All of the descendants of the original cell will therefore "remember" that the progenitor cell had experienced a transient signal that initiated the production of protein A.

The molecular mechanisms that create specialized cell types-The Expression of Different Genes Can Be Coordinated by a Single Protein Example: Figure 8-15: A single transcription regulator can coordinate the expression of many different genes.

*A single transcription regulator can coordinate the expression of many different genes.* The action of the cortisol receptor is illustrated. On the left is a series of genes, each of which has a different gene activator protein bound to its respective regulatory DNA sequences. However, these bound proteins are not sufficient on their own to activate transcription efficiently. On the right is shown the effect of adding an additional transcription regulator—the cortisol- receptor complex—that can bind to the same regulatory DNA sequence in each gene. The activated cortisol receptor completes the combination of transcription regulators required for efficient initiation of transcription, and the genes are now switched on as a set.

The molecular mechanisms that create specialized cell types- Figure 8-16: A small number of transcription regulators can convert one differentiated cell type directly into another.

*A small number of transcription regulators can convert one differentiated cell type directly into another.* In this experiment, liver cells grown in culture (A) were converted into neuronal cells (B) via the artificial introduction of three nerve-specific transcription regulators. The cells are labeled with a fluorescent dye.

How transcriptional switches work- Transcription Regulators Bind to Regulatory DNA Sequences Example: Figure 8-4: A transcription regulator interacts with the major groove of a DNA double helix.

*A transcription regulator interacts with the major groove of a DNA double helix.* (A) This regulator recognizes DNA via three α helices, shown as numbered cylinders, which allow the protein to fit into the major groove and form tight associations with the base pairs in a short stretch of DNA. This particular structural motif, called a homeodomain, is found in many eukaryotic DNA-binding proteins (Movie 8.1). (B) Most of the contacts with the DNA bases are made by helix 3 (red ), which is shown here end-on. The protein interacts with the edges of the nucleotides without disrupting the hydrogen bonds that hold the base pairs together. (C) An asparagine residue from helix 3 forms two hydrogen bonds with the adenine in an A-T base pair. The view is end-on looking down the DNA double helix, and the protein contacts the base pair from the major groove side. For simplicity, only one amino acid-base contact is shown; in reality, transcription regulators form hydrogen bonds (as shown here), ionic bonds, and hydrophobic interactions with individual bases in the major groove. Typically, the protein-DNA interface would consist of 10-20 such contacts, each involving a different amino acid and each contributing to the overall strength of the protein-DNA interaction.

The molecular mechanisms that create specialized cell types- Epigenetic Mechanisms Allow Differentiated Cells to Maintain Their Identity Example: Figure 8-21: Formation of 5-methylcytosine occurs by methylation of a cytosine base in the DNA double helix.

*Formation of 5-methylcytosine occurs by methylation of a cytosine base in the DNA double helix.* In vertebrates, this modification is confined to selected cytosine (C) nucleotides that fall next to a guanine (G) in the sequence CG.

The molecular mechanisms that create specialized cell types- Eukaryotic Genes Are Controlled by Combinations of Transcription Regulators Example: Figure 8-13: An experimental approach that involves the use of a reporter gene reveals the modular construction of the Eve gene regulatory region.

*An experimental approach that involves the use of a reporter gene reveals the modular construction of the Eve gene regulatory region.* (A) Expression of the Eve gene is controlled by a series of regulatory segments (orange) that direct the production of Eve protein in stripes along the embryo. (B) Embryos stained with antibodies to the Eve protein show the seven characteristic stripes of Eve expression. (C) In the laboratory, the regulatory segment that directs the formation of stripe 2 can be excised from the DNA shown in part A and inserted upstream of the E. coli LacZ gene, which encodes the enzyme β-galactosidase (see Figure 8-9). (D) When the engineered DNA containing the stripe 2 regulatory segment is introduced into the genome of a fly, the resulting embryo expresses β galactosidase precisely in the position of the second Eve stripe. Enzyme activity is assayed by the addition of X-gal, a modified sugar that when cleaved by β galactosidase generates an insoluble blue product.

Post-transcriptional controls- MicroRNAs Direct the Destruction of Target mRNAs Example: Figure 8-25: An miRNA targets a complementary mRNA molecule for destruction.

*An miRNA targets a complementary mRNA molecule for destruction.* Each precursor miRNA transcript is processed to form a double stranded intermediate, which is further processed to form a mature, single stranded miRNA. This miRNA assembles with a set of proteins into a complex called RISC, which then searches for mRNAs that have a nucleotide sequence complementary to its bound miRNA. Depending on how extensive the region of complementarity is, the target mRNA is either rapidly degraded by a nuclease within the RISC or transferred to an area of the cytoplasm where other cellular nucleases destroy it.

The molecular mechanisms that create specialized cell types- Figure 8-19: Artificially induced expression of the Drosophila Ey gene in the precursor cells of the leg triggers the misplaced development of an eye on a fly's leg

*Artificially induced expression of the Drosophila Ey gene in the precursor cells of the leg triggers the misplaced development of an eye on a fly's leg.*

The molecular mechanisms that create specialized cell types- Combinatorial Control Can Also Generate Different Cell Types Example: Figure 8-17: Combination of transcription factors can generate many cell types during development.

*Combination of transcription factors can generate many cell types during development.*

The molecular mechanisms that create specialized cell types- Figure 8-17: Combination of transcription factors can generate many cell types during development.

*Combinations of a few transcription regulators can generate many cell types during development.* In this simple scheme, a "decision" to make a new transcription regulator (shown as a numbered circle) is made after each cell division. Repetition of this simple rule can generate eight cell types (A through H), using only three transcription regulators. Each of these hypothetical cell types would then express many different genes, as dictated by the combination of transcription regulators that each cell type produces.

The molecular mechanisms that create specialized cell types- Epigenetic Mechanisms Allow Differentiated Cells to Maintain Their Identity Example: Figure 8-22: DNA methylation patterns can be faithfully inherited when a cell divides.

*DNA methylation patterns can be faithfully inherited when a cell divides.* An enzyme called a maintenance methyltransferase guarantees that once a pattern of DNA methylation has been established, it is inherited by newly made DNA. Immediately after DNA replication, each daughter double helix will contain one methylated DNA strand—inherited from the parent double helix—and one unmethylated, newly synthesized strand. The maintenance methyltransferase interacts with these hybrid double helices and methylates only those CG sequences that are base-paired with a CG sequence that is already methylated.

An overview of gene expression Figure 8-2a: Differentiated cells contain all the genetic instructions necessary to direct the formation of a complete organism.

*Differentiated cells contain all the genetic instructions necessary to direct the formation of a complete organism.* (A) The nucleus of a skin cell from an adult frog transplanted into an egg whose nucleus has been destroyed can give rise to an entire tadpole. The broken arrow indicates that to give the transplanted genome time to adjust to an embryonic environment, a further transfer step is required in which one of the nuclei is taken from the early embryo that begins to develop and is put back into a second enucleated egg.

An overview of gene expression Figure 8-2b: Differentiated cells contain all the genetic instructions necessary to direct the formation of a complete organism.

*Differentiated cells contain all the genetic instructions necessary to direct the formation of a complete organism.* (B) In many types of plants, differentiated cells retain the ability to "de-differentiate," so that a single cell can proliferate to form a clone of progeny cells that later give rise to an entire plant.

An overview of gene expression Figure 8-2c: Differentiated cells contain all the genetic instructions necessary to direct the formation of a complete organism.

*Differentiated cells contain all the genetic instructions necessary to direct the formation of a complete organism.* (C) A nucleus removed from a differentiated cell from an adult cow can be introduced into an enucleated egg from a different cow to give rise to a calf. Different calves produced from the same differentiated cell donor are all clones of the donor and are therefore genetically identical.

How transcriptional switches work- Eukaryotic Transcription Regulators Help Initiate Transcription by Recruiting Chromatin-Modifying Proteins Example: Figure 8-11: Eukaryotic transcriptional activators can recruit chromatin modifying proteins to help initiate gene transcription.

*Eukaryotic transcriptional activators can recruit chromatin modifying proteins to help initiate gene transcription.* On the right, chromatin remodeling complexes render the DNA packaged in chromatin more accessible to other proteins in the cell, including those required for transcription initiation; notice, for example, the increased exposure of the TATA box. On the left, the recruitment of histone modifying enzymes such as histone acetyltransferases adds acetyl groups to specific histones, which can then serve as binding sites for proteins that stimulate transcription initiation (not shown).

How transcriptional switches work- Eukaryotic Transcription Regulators Control Gene Expression from a Distance Example: Figure 8-10: In eukaryotes, gene DNA activation can occur at a distance.

*In eukaryotes, gene DNA activation can occur at a distance.* An activator protein bound to a distant enhancer attracts RNA polymerase and general transcription factors to the promoter. Looping of the intervening DNA permits contact between the activator and the transcription initiation complex bound to the promoter. In the case shown here, a large protein complex called Mediator serves as a go-between. The broken stretch of DNA signifies that the length of DNA between the enhancer and the start of transcription varies, sometimes reaching tens of thousands of nucleotide pairs in length. The TATA box is a DNA recognition sequence for the first general transcription factor that binds to the promoter (see Figure 7-12).

The molecular mechanisms that create specialized cell types- Combinatorial Control Can Also Generate Different Cell Types Example: Figure 8-16: In this experiment liver cells grown in culture were converted into neuronal cells via the artificial introduction of three nerve-specific transcription factors.

*In this experiment liver cells grown in culture were converted into neuronal cells via the artificial introduction of three nerve-specific transcription factors.*

How transcriptional switches work- An Activator and a Repressor Control the Lac Operon Example: Figure 8-9: The Lac operon is controlled by two transcription regulators, the Lac repressor and CAP.

*The Lac operon is controlled by two transcription regulators, the Lac repressor and CAP.* When lactose is absent, the Lac repressor binds to the Lac operator and shuts off expression of the operon. Addition of lactose increases the intracellular concentration of a related compound, allolactose; allolactose binds to the Lac repressor, causing it to undergo a conformational change that releases its grip on the operator DNA (not shown). When glucose is absent, cyclic AMP (red triangle) is produced by the cell, and CAP binds to DNA. LacZ, the first gene of the operon, encodes the enzyme β galactosidase, which breaks down lactose to galactose and glucose.

The molecular mechanisms that create specialized cell types- Eukaryotic Genes Are Controlled by Combinations of Transcription Regulators Example: Figure 8-14: The regulatory segment that specifies Eve stripe 2 contains binding sites for four different transcription regulators.

*The regulatory segment that specifies Eve stripe 2 contains binding sites for four different transcription regulators.* All four regulators are responsible for the proper expression of Eve in stripe 2. Flies that are deficient in the two activators, called Bicoid and Hunchback, fail to form stripe 2 efficiently; in flies deficient in either of the two repressors, called Giant and Krüppel, stripe 2 expands and covers an abnormally broad region of the embryo. As indicated in the diagram, in some cases the binding sites for the transcription regulators overlap, and the proteins compete for binding to the DNA. For example, the binding of Bicoid and Krüppel to the site at the far right is thought to be mutually exclusive. The regulatory segment is 480 base pairs in length.

The molecular mechanisms that create specialized cell types- Eukaryotic Genes Are Controlled by Combinations of Transcription Regulators Example: Figure 8-12: Transcription regulators work together as a "committee" to control the expression of a eukaryotic gene.

*Transcription regulators work together as a "committee" to control the expression of a eukaryotic gene.* Whereas the general transcription factors that assemble at the promoter are the same for all genes transcribed by RNA polymerase (see Figure 7-12), the transcription regulators and the locations of their DNA binding sites relative to the promoters are different for different genes. These regulators, along with chromatin modifying proteins, are assembled at the promoter by the Mediator. The effects of multiple transcription regulators combine to determine the final rate of transcription initiation.

Post-transcriptional controls- Small Interfering RNAs Are Produced From Double-Stranded, Foreign RNAs to Protect Cells From Infections Example: Figure 8-26: siRNAs are produced from double-stranded, foreign RNAs in the process of RNA interference.

*siRNAs are produced from double-stranded, foreign RNAs in the process of RNA interference.* Double stranded RNAs from a virus or transposable genetic element are first cleaved by a nuclease called Dicer. The resulting double stranded fragments are incorporated into RISCs, which discard one strand of the foreign RNA duplex and use the other strand to locate and destroy foreign RNAs with a complementary sequence.

Post-transcriptional controls

-Each mRNA Controls Its Own Degradation and Translation -Regulatory RNAs Control the Expression of Thousands of Genes -MicroRNAs Direct the Destruction of Target mRNAs -Small Interfering RNAs Are Produced From Double-Stranded, Foreign RNAs to Protect Cells From Infections -Thousands of Long Noncoding RNAs May Also Regulate Mammalian Gene Activity

The molecular mechanisms that create specialized cell types

-Eukaryotic Genes Are Controlled by Combinations of Transcription Regulators -The Expression of Different Genes Can Be Coordinated by a Single Protein -Combinatorial Control Can Also Generate Different Cell Types -Specialized Cell Types Can Be Experimentally Reprogrammed to Become Pluripotent Stem Cells -The Formation of an Entire Organ Can Be Triggered by a Single Transcription Regulator -Epigenetic Mechanisms Allow Differentiated Cells to Maintain Their Identity

How transcriptional switches work- Transcription Regulators Bind to Regulatory DNA Sequences Example: Figure 8-5: Many transcription regulators bind to DNA as dimers.

-Many transcription factors bind to DNA as dimers. *Many transcription regulators bind to DNA as dimers.* This transcription regulator contains a leucine zipper motif, which is formed by two α helices, each contributed by a different protein subunit. Leucine zipper proteins thus bind to DNA as dimers, gripping the double helix like a clothespin on a clothesline (Movie 8.2).

An overview of gene expression

-The Different Cell Types of a Multicellular Organism Contain the Same DNA -Different Cell Types Produce Different Sets of Proteins -A Cell Can Change the Expression of Its Genes in Response to External Signals -Gene Expression Can Be Regulated at Various Steps from DNA to RNA to Protein

An overview of gene expression- Different cell types of multicellular organism contain the same DNA Example: Figure 8-2b: Differentiated cells contain all the genetic instructions necessary to direct the formation of a complete organism.

De-differentiation (1-4) Tightly controlled development (5-8)

If these two cells came from the same human - do the neuron and liver cell contain the same genome? If yes, how is this possible?

Different expression of genes. Over 98% of genome between chimpanzee & human are similar; why do they look so different? Because of when and where genes are expressed, esp during development. - Spatial (where) & temporal (time) expression.

The molecular mechanisms that create specialized cell types- The Formation of an Entire Organ Can Be Triggered by a Single Transcription Regulator Example: Figure 8-19: Artificially induced expression of the Drosophila Ey gene in the precursor cells of the leg triggers the misplaced development of an eye on a fly's leg

Ectopic expression of the Ey gene in developing tissue that will ultimately form a leg.

How transcriptional switches work- An Activator and a Repressor Control the Lac Operon

Glucose inhibits adenyl cyclase function.

An overview of gene expression- Different cell types of multicellular organism contain the same DNA Example: Figure 8-2a: Differentiated cells contain all the genetic instructions necessary to direct the formation of a complete organism.

Tightly controlled development

An overview of gene expression- Different cell types of multicellular organism contain the same DNA Example: Figure 8-2c: Differentiated cells contain all the genetic instructions necessary to direct the formation of a complete organism.

Tightly controlled development