Gene Regulation Objectives: Understand how both prokaryotes and eukaryotes control the expression of genes

GENE REGULATION Virtually every cell in your body contains a complete set of genes But they are not all turned on in every tissue Each cell in your body expresses only a small subset of genes at any time During development different cells express different sets of genes in a precisely regulated fashion THIS IS A GOOD THING BECAUSE YOU DO NOT WANT HAIR TO GROW ON YOUR TONGUE OR FINGERNAILS ON YOUR FACE WE HAVE TOUCHED ON POLYGENIC TRATIS AND THOSE WITH GENETIC AND EVIRONMENTAL COMPONENTS NOW WE ARE GOING TO LOOK A BIT AT HOW DOES A CELL DECIDE WHAT KIND OF CELL IT WILL BE WHAT REGULATES WHAT GENES WILL BE TURNED ON AND OFF

Differentiation involves cell specialization, in both structure and function Differentiation is controlled by turning specific sets of genes on or off, NOT by differences in DNA between cells For the BLAST Animation Signaling Across Membranes, go to Animation and Video Files. Student Misconceptions and Concerns 1. The broad concept of selective reading of the genetic code associated with differentiation and types of cellular activity can be missed when concentrating on the extensive details of regulation. Analogies, noted below in the teaching tips, can help students relate this overall selective process to their own experiences. Students already understand the selective reading of relevant chapters in textbooks and the selective referencing of software manuals to get answers to different questions. These experiences are similar in many ways to the broad processes of gene regulation. 2. The many levels of gene regulation in eukaryotic cells can be confusing and frustrating. The water pipe analogy depicted in Figure 11.9 can be a helpful reference to organize the potential sites of regulation. Teaching Tips 1. The control of gene expression is analogous to buying a book about how to build birdhouses and reading only the plans needed to build one particular model. Although the book contains directions to build many different birdhouses, you read and follow only the directions for the particular birdhouse you choose to build. The pages and directions for the other birdhouses remain intact. When cells differentiate, they read, or express, only the genes that are needed in that particular cell type. Copyright © 2009 Pearson Education, Inc.

GENE REGULATION Gene regulation occurs at the level of transcription or production of mRNA Occurs VERY differently in prokaryotes and in eukaryotes A given cell transcribes only a specific set of genes and not others WHAT IS MRNA? THAT IS WHY WE SPENT SO MUCH TIME ON HOW PROTEINS ARE MADE REVIEW HAVE THE INSULIN GENE IN ALL YOUR CELLS BUT IT IS ONLY EXPRESSED OR TRANSCRIBED BY CELLS IN THE PANCREASE NOT BY SKIN CELLS

CENTRAL DOGMA Genetic information always goes from DNA to RNA to protein Gene regulation has been well studied in E. coli, especially metabolic genes When a bacterial cell’s food changes it will alter the manufacturing of the enzymes necessary to metabolize that food WHY E COLI? What do you like to eat? WHEN YOU EAT YOU NEED ENZYMES TO BREAK DOWN THE FOOD YOU EAT EXPLAIN WHAT ENZYMES ARE THEY BREAK DOWN FOOD LIKE GLUCOSE AND LACTOSE WHAT ARE GLUCOSE AND LACTOSE?

Prokaryotic Gene Regulation An operon is a group of genes under coordinated control in bacteria Promoter sequence where RNA polymerase binds Operator sequence is where a repressor can bind and block RNA polymerase action The actual gene/genes that make an enzyme Teaching Tips 1. The lactose operon is turned on by removing the repressor . . . a sort of double negative. Students might enjoy various analogies to other situations, including the familiar refrain “When the cat's away, the mice will play.” In another analogy, if Mom keeps the kids away from the cookies, but somebody occupies her attention, kids can sneak by and snatch some cookies. In this analogy, the person occupying Mom’s attention functions most like lactose binding to the repressor. 2. A key advantage of an operon system is the ability to turn off or on a set of genes with a single “switch.” You can demonstrate this relationship in your classroom by turning off or on a set of lights with a single switch. 3. The control of gene expression is analogous to buying a book about how to build birdhouses and reading only the plans needed to build one particular model. Although the book contains directions to build many different birdhouses, you read and follow only the directions for the particular birdhouse you choose to build. The pages and directions for the other birdhouses remain intact. When cells differentiate, they read, or express, only the genes that are needed in that particular cell type. Copyright © 2009 Pearson Education, Inc.

Prokaryotic Gene Regulation Types of operon control Inducible operon – usually OFF Takes advantage of an unusual situation Active repressor binds to the operator Inducer binds to and inactivates the repressor The lac operon has an activator called catabolite activator protein (CAP). CAP responds to levels of glucose, the main energy source for the cell. If glucose supplies are abundant, CAP is inactive. If glucose levels fall, cyclic AMP is produced as a by-product of the declining energy levels. Cyclic AMP binds to and activates CAP protein. Activated CAP protein binds to the promoter region and enhances the binding of RNA polymerase, increasing the rate of transcription of the lactose-utilization genes. The following table summarizes lac operon control with various levels of lactose and glucose present. Student Misconceptions and Concerns 1. The broad concept of selective reading of the genetic code associated with differentiation and types of cellular activity can be missed when concentrating on the extensive details of regulation. Analogies, noted below in the teaching tips, can help students relate this overall selective process to their own experiences. Students already understand the selective reading of relevant chapters in textbooks and the selective referencing of software manuals to get answers to different questions. These experiences are similar in many ways to the broad processes of gene regulation. 2. The many levels of gene regulation in eukaryotic cells can be confusing and frustrating. The water pipe analogy depicted in Figure 11.9 can be a helpful reference to organize the potential sites of regulation. Teaching Tips 1. The lactose operon is turned on by removing the repressor . . . a sort of double negative. Students might enjoy various analogies to other situations, including the familiar refrain “When the cat's away, the mice will play.” In another analogy, if Mom keeps the kids away from the cookies, but somebody occupies her attention, kids can sneak by and snatch some cookies. In this analogy, the person occupying Mom’s attention functions most like lactose binding to the repressor. 2. A key advantage of an operon system is the ability to turn off or on a set of genes with a single “switch.” You can demonstrate this relationship in your classroom by turning off or on a set of lights with a single switch. 3. The control of gene expression is analogous to buying a book about how to build birdhouses and reading only the plans needed to build one particular model. Although the book contains directions to build many different birdhouses, you read and follow only the directions for the particular birdhouse you choose to build. The pages and directions for the other birdhouses remain intact. When cells differentiate, they read, or express, only the genes that are needed in that particular cell type. Copyright © 2009 Pearson Education, Inc.

Figure 11.1A Cells of E.coli bacteria.

Lactose-utilization genes OPERON Regulatory gene Promoter Operator Lactose-utilization genes DNA mRNA RNA polymerase cannot attach to promoter Active repressor Protein Figure 11.1B The lac operon. The lac operon is inducible, usually off, unless the inducer, lactose, is present. In the absence of lactose, active repressor binds to the operator and prevents transcription from the promoter sequence. It should be noted that small amounts of repressor are always present in the cell, since there is no operator region between the regulatory gene promoter (not shown) and the regulatory gene. The repressor binds reversibly to the operator region upstream of the lactose-utilization enzyme genes. When repressor is bound, RNA polymerase cannot initiate transcription from the promoter region. If lactose is present in the environment, some can diffuse into the cell and bind to free repressors, inactivating them. Inactivated repressors cannot bind to the operator region. Therefore, RNA polymerase can bind to the promoter and initiate transcription of the lactose-utilization enzyme genes. An mRNA with coding regions for all three genes is produced and then three separate proteins are translated from this mRNA. These proteins allow the cell to use lactose as an energy source. Operon turned off (lactose absent)

Operon turned on (lactose inactivates repressor) DNA RNA polymerase bound to promoter mRNA Protein Enzymes for lactose utilization Lactose Inactive repressor Figure 11.1B The lac operon. The lac operon is inducible, usually off, unless the inducer, lactose, is present. In the absence of lactose, active repressor binds to the operator and prevents transcription from the promoter sequence. It should be noted that small amounts of repressor are always present in the cell, since there is no operator region between the regulatory gene promoter (not shown) and the regulatory gene. The repressor binds reversibly to the operator region upstream of the lactose-utilization enzyme genes. When repressor is bound, RNA polymerase cannot initiate transcription from the promoter region. If lactose is present in the environment, some can diffuse into the cell and bind to free repressors, inactivating them. Inactivated repressors cannot bind to the operator region. Therefore, RNA polymerase can bind to the promoter and initiate transcription of the lactose-utilization enzyme genes. An mRNA with coding regions for all three genes is produced and then three separate proteins are translated from this mRNA. These proteins allow the cell to use lactose as an energy source. Operon turned on (lactose inactivates repressor)

Prokaryotic Gene Regulation Types of operon control Repressible operon – usually ON (trp operon) Usually produces a necessary product not encountered in environment Repressor is initially inactive Co-repressor (tryptophan) binds to the repressor and makes it active For many operons, activators enhance RNA polymerase binding to the promoter The lac operon has an activator called catabolite activator protein (CAP). CAP responds to levels of glucose, the main energy source for the cell. If glucose supplies are abundant, CAP is inactive. If glucose levels fall, cyclic AMP is produced as a by-product of the declining energy levels. Cyclic AMP binds to and activates CAP protein. Activated CAP protein binds to the promoter region and enhances the binding of RNA polymerase, increasing the rate of transcription of the lactose-utilization genes. The following table summarizes lac operon control with various levels of lactose and glucose present. Student Misconceptions and Concerns 1. The broad concept of selective reading of the genetic code associated with differentiation and types of cellular activity can be missed when concentrating on the extensive details of regulation. Analogies, noted below in the teaching tips, can help students relate this overall selective process to their own experiences. Students already understand the selective reading of relevant chapters in textbooks and the selective referencing of software manuals to get answers to different questions. These experiences are similar in many ways to the broad processes of gene regulation. 2. The many levels of gene regulation in eukaryotic cells can be confusing and frustrating. The water pipe analogy depicted in Figure 11.9 can be a helpful reference to organize the potential sites of regulation. Teaching Tips 1. The lactose operon is turned on by removing the repressor . . . a sort of double negative. Students might enjoy various analogies to other situations, including the familiar refrain “When the cat's away, the mice will play.” In another analogy, if Mom keeps the kids away from the cookies, but somebody occupies her attention, kids can sneak by and snatch some cookies. In this analogy, the person occupying Mom’s attention functions most like lactose binding to the repressor. 2. A key advantage of an operon system is the ability to turn off or on a set of genes with a single “switch.” You can demonstrate this relationship in your classroom by turning off or on a set of lights with a single switch. 3. The control of gene expression is analogous to buying a book about how to build birdhouses and reading only the plans needed to build one particular model. Although the book contains directions to build many different birdhouses, you read and follow only the directions for the particular birdhouse you choose to build. The pages and directions for the other birdhouses remain intact. When cells differentiate, they read, or express, only the genes that are needed in that particular cell type. Copyright © 2009 Pearson Education, Inc.

Left Page: Operon vehicles Operons tend to either block RNA Polymerase from binding to the DNA, or help it. Your job is to design a vehicle (and name it) based off of an operon. You must include the name, what the vehicle looks like (4 colors), which operon it is based on, what abilities the vehicle has (based off of the operon), and how you would convince someone to buy it.

Eukaryotic Gene Regulation In eukaryotic organisms like ourselves there are several methods of regulating protein production DNA/Gene packaging Transciption controls (promoters, activators, enhancers) Alternative RNA splicing

DNA/Gene Packaging Eukaryotic chromosomes undergo multiple levels of folding and coiling, called DNA packing DNA is wrapped around proteins “Beads on a string” appearance Supercoil is a coiling of the tight helical fiber DNA packing can prevent transcription

Transcription Controls Similar to prokaryotic gene regulation Genes are controlled by regulatory elements in the promoter region that act like on/off or dimmer switches Regulatory proteins that bind to control sequences Transcription factors promote RNA polymerase binding to the promoter Activator proteins bind to DNA enhancers and interact with other transcription factors

Enhancers Promoter Gene DNA Activator proteins Transcription factors Gene DNA Activator proteins Transcription factors Other proteins RNA polymerase Figure 11.5 A model for the turning on of a eukaryotic gene. Sequence of events: Activator proteins bind to an enhancer sequence. DNA bends to bring the enhancer sequence closer to the promoter region. Activators interact with other transcription factors that bind to the promoter. RNA polymerase is properly positioned on the promoter and transcription is initiated. Bending of DNA Transcription

Alternative RNA Splicing Eukaryotic DNA differs from prokaryotic DNA in that the coding sequences along the gene are interspersed with noncoding sequences The coding sequences are called EXONS The non coding sequences are called INTRONS (they INterfere with gene)

Alternative RNA Splicing After the initial transcript is produced the introns are spliced out to form the completed message ready for translation Introns can be very large and numerous, so some genes are much bigger than the final processed mRNA

Alternative RNA Splicing Exons can be spliced together in different ways This allows a variety of different polypeptides to be assembled from the same gene Alternate splicing is common in insects and vertebrates, where 2 or 3 different proteins are produced from one gene – esp. for immune system products Teaching Tips 1. Alternative RNA splicing is like remixing music to produce a new song or re-editing a movie for a different effect.

Exons DNA 1 2 3 4 5 RNA transcript 1 2 3 4 5 RNA splicing or mRNA 1 2 Exons DNA 1 2 3 4 5 RNA transcript 1 2 3 4 5 Figure 11.6 Production of two different mRNAs from the same gene. This figure shows exon skipping, one mode of alternative splicing. One product contains exon 3 and involved the removal of exon 4 with introns on either side. This splicing start site (5) was on the intron between 3 and 4 and the splicing end site (3) was on the intron between 4 and 5. The other product contains exon 4 and involved the removal of exon 3 with the introns on either side. RNA splicing or mRNA 1 2 3 5 1 2 4 5

Differentiated Cells Most differentiated cells retain a full set of genes, even though only a subset may be expressed Plant cloning - A root cell can divide to form an adult plant Animal limb regeneration (i.e. starfish) De-differentiation followed by re-differentiation into specialized cells Student Misconceptions and Concerns 1. Students often fail to see the similarities between identical twins and cloning. Each process produces multiple individuals with identical nuclear genetic material. 2. Students often assume that clones will appear and act identically. This misunderstanding provides an opportunity to discuss the important influence of the environment in shaping the final phenotype. Teaching Tips 1. The basic question asked in Module 11.14 is whether a cell becomes differentiated by selectively reading the genome or by retaining only the needed sections. In your course, you are unlikely to assign the entire Concepts textbook. Instead, you likely ask your students to selectively read chapters in the book. Students could remove all of the pages that they do not need, leaving only those that you have assigned. Students that keep their textbooks intact, reading only the assigned and relevant passages, behave like cells undergoing differentiation. 2. An even more remarkable aspect of salamander limb regeneration is that only the missing limb segments are regenerated. If an arm is amputated at the elbow, only the forearm, wrist, and hand are regenerated. Somehow, the cells can detect what is missing and replace only those parts! Copyright © 2009 Pearson Education, Inc.

Cell division in culture Root of carrot plant Single cell Figure 11.14 Growth of a carrot plant from a differentiated root cell. This figure shows how an entire plant can be produced from a single root cell. Root cells cultured in nutrient medium Cell division in culture Plantlet Adult plant

Left Page Assignment: Answer the two questions below: If all of the DNA in all of an individual’s cells are the same, how is it possible to have different cell types? How is it possible that the cells lining your stomach and the cells of your retina (eyeball) have identical DNA in their nuclei?

Pre-AP Bio I will buy the class that achieves the highest class average on the test on Friday donuts. So study hard!