Regulation of Gene Expression Chapter 18

Overview of Gene Expression Regulation of Gene Expression Overview of Gene Expression The control of gene expression is vital to the proper and efficient functioning of an organism. Cells control metabolism by either regulating enzyme activity –or- regulating the expression of genes coding for enzymes.

Figure 18.2 Figure 18.2 – Regulation of a Metabolic Pathway In this example of the pathway of tryptophan synthesis, an abundance of tryptophan can either Inhibit the activity of the first enzyme in the pathway (feedback inhibition) Repress expression of the genes encoding all subunits of the enzymes in the pathway

Control of Gene Expression in Bacteria Prokaryotic Gene Regulation Control of Gene Expression in Bacteria Bacteria often respond to environmental change by regulating transcription. In bacteria, genes are often clustered into operons, with one promoter serving several adjacent genes. An operator site on the DNA switches the operon on or off, resulting in coordinate regulation of the genes.

Operons: The Basic Concept Prokaryotic Gene Regulation Operons: The Basic Concept An operon is essentially a set of genes and the switches that control the expression of those genes. An operon consists of: operator promotor and genes that they control All together, the operator, the promoter, and the genes they control – the entire stretch of DNA required for enzyme production for the pathway – is called an operon.

Prokaryotic Gene Regulation The Operon Model Promoter Region: a base sequence that signals the start site for gene transcription; this is where RNA polymerase binds to begin the process. Operator: a short sequence near the promoter that assists in transcription by interacting with regulatory proteins (transcription factors). Repressor: protein that prevents the binding of RNA polymerase to the promoter site. Enhancer: DNA region, also known as “regulator”, that is located thousands of bases away from the promoter; it influences transcription by interacting with specific transcription factors. Inducer: a molecule that binds to and inactivates a repressor (i.e. lactose for the lac operon). Operon: a promoter/operator pair that services multiple genes.

Repressible & Inducible Operons Prokaryotic Gene Regulation Repressible & Inducible Operons There are basically two types of operons found in prokaryotes: repressible operons and inducible operons. Both the repressible and inducible operon are types of NEGATIVE gene regulation because both are turned OFF by the active form of the repressor protein. In either type of operon, binding of a specific repressor protein to the operator shuts off transcription. Trp operon – repressible operon is always in the on position until it is not needed and becomes repressed or switched off. Lac operon – inducible operon is always off until it is induced to turn on.

Figure 18.3a – The trp Operon Tryptophan is an amino acid produced by an anabolic pathway catalyzed by repressible enzymes. If tryptophan is absent, the repressor is inactive, the operon is on, and RNA polymerase attaches to the DNA at the promoter and transcribes the operon’s genes. http://bcs.whfreeman.com/thelifewire/content/chp13/1302002.html

Figure 18.3b – The trp Operon As tryptophan accumulates, it inhibits its own production by activating the repressor protein. The repressor switches the operon off by binding to the operator and blocking access of RNA polymerase to the promoter. Tryptophan binds to an allosteric site on the protein, causing its conformation to change to the active form. REMEMBER: The trp operon is an example of a repressible operon because it is always in the ON position until not needed – then it is switched off. http://highered.mcgraw-hill.com/olc/dl/120080/bio26.swf

Figure 18.4a – The lac Operon The lactose operon services a series of three genes involved in the process of lactose metabolism. These genes help bacteria to digest lactose. It makes sense for these bacteria to express these genes only when lactose is present…otherwise, they waste energy on unneeded enzymes. This is where OPERONS come in to play…the lac repressor is innately active, and in the absence of lactose it switches off the operon by binding to the operator on the promoter region and blocking transcription from occurring. http://www.sumanasinc.com/webcontent/animations/content/lacoperon.html

Figure 18.4b – The lac Operon When lactose is present, there is a binding site on the repressor where lactose attaches, causing the repressor to let go of the promoter region. RNA polymerase is then free to bind to that site and initiate transcription of the genes. IN THIS EXAMPLE…allolactose, an isomer of lactose, de-represses the operon by inactivating the repressor. In this way, the enzymes for lactose utilization are induced. REMEMBER: the lac operon is an example of an inducible operon because it is always off until it is induced to turn on. http://highered.mcgraw-hill.com/sites/dl/free/0072835125/126997/animation27.html

Positive Gene Regulation Prokaryotic Gene Regulation Positive Gene Regulation When glucose and lactose are both present in its environment, E. coli prefer to use glucose. Only when lactose is present AND glucose is in short supply does E. coli use lactose as an energy source, and only then does it synthesize appreciable quantities of the enzymes for lactose breakdown. How does the E. coli cell sense the glucose concentration and relay this information to its genome? http://highered.mcgraw-hill.com/olc/dl/120080/bio27.swf

Figure 18.5a – Positive Control If glucose is scarce, the high level of cAMP activates CAP, and the lac operon produces large amounts of mRNA coding for the enzymes in the lactose pathway.

Figure 18.5b – Positive Control When glucose is present, cAMP is scarce, and CAP is unable to stimulate transcription.

Factors Affecting Ability of Repressor to Bind to Operator Prokaryotic Gene Regulation Factors Affecting Ability of Repressor to Bind to Operator Co-Repressor : Activates a Repressor Seen in the trp Operon Co-Repressor is tryptophan Turns normally “on” Operon “off” Inducer: Inactivates a Repressor, Induces the Gene to be Transcribed Seen in the lac Operon Inducer is allolactose Turns normally “off” Operon “on” 15

Review: Structure/Function of Prokaryotic Chromosomes Prokaryotic Gene Regulation Review: Structure/Function of Prokaryotic Chromosomes shape (circular/nonlinear/loop) less complex than eukaryotes (no histones/less elaborate structure/folding) size (smaller size/less genetic information/fewer genes) replication method (single origin of replication/rolling circle replication) transcription/translation may be coupled generally few or no introns (noncoding segments) majority of genome expressed operons are used for gene regulation and control NOTE: plasmids – more common but not unique to prokaryotes/not part of prokaryote chromosome

The Structure of the Chromosome Chromosome Structure The Structure of the Chromosome In Prokaryotes: The bacterial chromosome is a double-stranded, circular DNA molecule associated with a small amount of protein In a bacterium, the DNA is “supercoiled” and found in a region of the cell called the nucleoid In Eukaryotes: Eukaryotic chromosomes have linear DNA molecules associated with a large amount of protein Chromatin is a complex of DNA and protein, and is found in the nucleus of eukaryotic cells Histones are proteins that are responsible for the first level of DNA packing in chromatin

Chromosome Structure of Eukaryotes Eukaryotic Chromosomes Chromosome Structure of Eukaryotes Most chromatin is loosely packed in the nucleus during interphase and condenses into chromosomes prior to mitosis. Loosely packed chromatin is called euchromatin. During interphase a few regions of chromatin (centromeres and telomeres) are highly condensed into heterochromatin. Dense packing of the heterochromatin makes it difficult for the cell to express genetic information coded in these regions. Eukaryotic chromosomes contain DNA wrapped around proteins called histones. The strands of nucleosomes are tightly coiled and supercoiled to form chromosomes.

Eukaryotic Chromosomes Figure 16.21b Chromatin packing in a eukaryotic chromosome Chromatin is organized into fibers 10-nm fiber DNA winds around histones to form nucleosome “beads” Nucleosomes are strung together like beads on a string by linker DNA 30-nm fiber Interactions between nucleosomes cause the thin fiber to coil or fold into this thicker fiber 300-nm fiber The 30-nm fiber forms looped domains that attach to proteins Metaphase chromosome The looped domains coil further The width of a chromatid is 700 nm

Control of Gene Expression in Eukaryotes Eukaryotic Gene Regulation Control of Gene Expression in Eukaryotes Eukaryotic gene expression can be regulated at any stage. Because gene expression in eukaryotes involves more steps, there are more places where gene control can occur. Opportunities for the control of gene expression in eukaryotes include: Chromatin Packing, modification Assembling of Transcription Factors RNA Processing Regulation of mRNA degradation and Control of Translation Protein Processing and Degradation

Overview Figure 18.6 THIS FIGURE IS HIGHLIGHTING KEY STAGES IN THE EXPRESSION OF A PROTEIN-CODING GENE. The expression of a given gene will not necessarily involve every stage shown. MAIN LESSON: each stage is a potential control point where gene expression can be turned on or off, sped up, or slowed down.

Expression of Genes in Eukaryotes Eukaryotic Gene Regulation Expression of Genes in Eukaryotes Eukaryotic cells face the same challenges as prokaryotic cells in expressing their genes, but with two main differences: The much greater size of the typical eukaryotic genome; importance of cell specialization in multicellular eukaryotes. In both prokaryotes and eukaryotes, DNA associates with proteins to form chromatin, but in the eukaryotic cell, the chromatin is ordered into higher structural levels.

Eukaryotic Chromosome Structure Eukaryotic Gene Regulation Eukaryotic Chromosome Structure Chromatin structure is based on successive levels of DNA packing. Eukaryotic chromatin is composed mostly of DNA and histone proteins that bind to the DNA to form nucleosomes, the most basic units of DNA packing. Additional folding leads ultimately to highly compacted heterochromatin, the form of chromatin in a metaphase chromosome. In interphase cells, most chromatin is in a highly extended form, called euchromatin. Chromatin structure is based on successive levels of DNA packing. Chromatin = term for DNA + Proteins in the Nucleus of a Eukaryotic cell DNA is wound around Histones (positively charged proteins which attract negatively charged DNA) Histones (8) with wound DNA make up a Nucleosome - the fundamental packing unit of Chromosome (10nm fiber) Nucleosomes further condense and form 30nm Chromatin Fibers Nucleosomes scaffold, loop, fold to make Looped Domains (300nm – 700nm fibers) Metaphase Chromosome – most condensed, largest form of DNA (1400nm) 23

Eukaryotic Gene Regulation The Eukaryotic Genome In prokaryotes, most of the DNA in a genome codes for protein, with a small amount of noncoding DNA that consists mainly of regulatory sequences such as promoters. In eukaryotic genomes, most of the DNA (97% in humans) does NOT encode protein or RNA. This DNA includes introns and repetitive DNA: Repetitive DNA are nucleotide sequences that are present in many copies in a genome, usually not within genes.

Chromatin Modifications Eukaryotic Gene Regulation Chromatin Modifications Chromatin modifications affect the availability of genes for transcription: The physical state of DNA in or near a gene is important in helping control whether the gene is available for transcription. Genes of heterochromatin (highly condensed) are usually not expressed because transcription proteins cannot reach the DNA. DNA methylation seems to diminish transcription of that DNA. Histone acetylation seems to loosen nucleosome structure and thereby enhance transcription.

Eukaryotic Gene Regulation DNA Methylation DNA methylation is the attachment of methyl groups (-CH3) to DNA bases after DNA is synthesized. Methylation renders DNA inactive. Inactive DNA, such as that of inactivated mammalian X chromosomes (Barr bodies), is generally highly methylated compared to DNA that is actively transcribed. Comparison of the same genes in different types of tissues shows that the genes are usually more heavily methylated in cells where they are not expressed. In addition, de-methylating certain inactive genes (removing their extra methyl groups) turns them on. At least in some species, DNA methylation seems to be essential for the long-term inactivation of genes that occurs during cellular differentiation in the embryo.

Eukaryotic Gene Regulation Histone Acetylation Histone acetylation is the attachment of acetyl groups (-COOH3) to certain amino acids of histone proteins; de-acetylation is the removal of acetyl groups. When the histones of nucleosome are acetylated, they change shape so that they grip the DNA less tightly. As a result, transcription proteins have easier access to genes in the acetylated region.

Transcription Initiation Eukaryotic Gene Regulation Transcription Initiation Transcription is controlled by the presence or absence of particular transcription factors, which bind to the DNA and affect the rate of transcription. Thus…transcription initiation is controlled by proteins that interact with DNA and with each other. Once a gene is “unpacked”, the initiation of transcription is the most important and universally used control point in gene expression.

Eukaryotic Gene and its Transcript Figure 18.8 Eukaryotic Gene and its Transcript REMEMBER: Transcription is controlled by the presence or absence of particular transcription factors, which bind to the DNA and affect the rate of transcription.

Assembling of Transcription Factors Activator proteins bind to enhancer sequences in the DNA and help position the initiation complex on the promoter. DNA bending brings the bound activators closer to the promoter. Other transcription factors and RNA polymerase are nearby. Protein-binding domains on the activators attach to certain transcription factors and help them form an active transcription initiation complex on the promoter. http://highered.mcgraw-hill.com/olc/dl/120080/bio28.swf Control elements are simply segments of noncoding DNA that help regulate transcription of a gene by binding proteins (transcription factors). 30

Post-Transcriptional Factors Eukaryotic Gene Regulation Post-Transcriptional Factors Transcription alone DOES NOT constitute gene expression! Post-transcriptional mechanisms play supporting roles in the control of gene expression: Alternative RNA splicing – where different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns. Regulatory proteins specific to a cell type control intron-exon choices by binding to regulatory sequences within the primary transcript. http://highered.mcgraw-hill.com/olc/dl/120080/bio31.swf

Alternative Splicing Offers New Combinations of Exons = New Proteins Eukaryotic Gene Regulation Alternative Splicing Offers New Combinations of Exons = New Proteins The RNA transcripts of some genes can be spliced in more than one way, generating different mRNA molecules. With alternative splicing, an organism can get more than one type of polypeptide from a single gene. 32

Further Control of Gene Expression Eukaryotic Gene Regulation Further Control of Gene Expression After RNA processing, other stages of gene expression that the cell may regulate are mRNA degradation, translation initiation, and protein processing and degradation. The life span of mRNA molecules in the cytoplasm is an important factor in determining the pattern of protein synthesis in a cell. Most translational control mechanisms block the initiation stage of polypeptide synthesis, when ribosomal subunits and the initiator tRNA attach to an mRNA.

Protein Processing and Degradation Eukaryotic Gene Regulation Protein Processing and Degradation The final opportunities for controlling gene expression occur after translation: Protein processing – cleavage and the addition of chemical groups required for function. Transport of the polypeptide to targeted destinations in the cell. Cells can also limit the lifetimes of normal proteins by selective degradation – chopped up by proteasomes.

Overview Figure 18.6 THIS FIGURE IS HIGHLIGHTING KEY STAGES IN THE EXPRESSION OF A PROTEIN-CODING GENE. The expression of a given gene will not necessarily involve every stage shown. MAIN LESSON: each stage is a potential control point where gene expression can be turned on or off, sped up, or slowed down.

The Molecular Biology of Cancer The Biology of Cancer The Molecular Biology of Cancer Certain genes normally regulate growth and division – the cell cycle – and mutations that alter those genes in somatic cells can lead to cancer. Proto-Oncogenes are normal genes that code for proteins which stimulate normal cell growth and division. Oncogenes – cancer causing genes; lead to abnormal stimulation of cell cycle. Oncogenes arise from genetic changes in proto-oncogenes: Amplification of proto-oncogenes Point mutation in proto-oncogene Movement of DNA within genome

The Biology of Cancer Genetic Changes Can Turn Proto-oncogenes into Oncogenes http://www.learner.org/courses/biology/units/cancer/images.html Proto-oncogenes can be converted to oncogenes by Movement of DNA within the genome: if it ends up near an active promoter, transcription may increase Amplification of a proto-oncogene: increases the number of copies of the gene Point mutations in the proto-oncogene or its control elements: causes an increase in gene expression   37

Tumor-Suppressor Genes The Biology of Cancer Tumor-Suppressor Genes In addition to mutations affecting growth-stimulating proteins, changes in genes whose normal products INHIBIT cell division also contribute to cancer: Such genes are called tumor-suppressor genes because the proteins they encode normally help prevent uncontrolled cell growth.

The Biology of Cancer p53 Tumor Suppressor and ras Proto-Oncogenes http://www.learner.org/courses/biology/units/cancer/images.html Mutations in the p53 tumor-suppressor gene and the ras proto-oncogene are very common in human cancers. Both are components of signal-transduction pathways that convey external signal to the DNA in the cell’s nucleus. Product of ras gene is G Protein (relays a growth signal and stimulates cell cycle). An oncogene protein that is a hyperactive version of this protein in the pathway can increase cell division. P53 protein – “guardian angel of the genome” DNA damage (UV, toxins) signals expression of p53 and p53 protein acts as transcription factor for gene p21 p21 halts cell cycle, allowing DNA repair P53 also can cause ‘cell suicide’ if damage is too great Many cancer patients p53 gene product does not function properly!

Figure 18.21 Signaling pathways that regulate cell growth (Layer 2) RAS and P53 contribute to uninhibited cell stimulation and growth- Tumor Formation Figure 18.21 Signaling pathways that regulate cell growth (Layer 2) 40

The Biology of Cancer Figure 18.22 A multi-step model for the development of colorectal cancer 41

Review: Structure/Function of Eukaryotic Chromosomes Chromatids 2/sister/pari/identical DNA/ genetic information distribution of one copy to each new cell Centromere noncoding/uncoiled/narrow/constricted region joins/holds/attaches chromatids together Nucelosome histones/DNA wrapped arround special proteins packaging compacting Chromatin Form (heterochromatin/euchromatin) heterochromatin is condensed/supercoiled proper distribution in cell division (not during replication) euchromatin is loosely coiled gene expression during interphase/replication occurs when loosely packed Kinetochores disc-shaped proteins spindle attachment/alignment Genes or DNA brief DNA description codes for proteins or for RNA Telomeres tips, ends, noncoding repetitive sequences protection against degradation/ aging, limits number of cell divisions

USEFUL ANIMATIONS http://highered.mcgraw-hill.com/olc/dl/120080/bio31.swf http://highered.mcgraw-hill.com/olc/dl/120077/bio25.swf http://highered.mcgraw-hill.com/olc/dl/120080/bio28.swf http://highered.mcgraw-hill.com/olc/dl/120082/bio34b.swf http://www.learner.org/courses/biology/units/cancer/images.html

You should now be able to: NEED TO KNOW You should now be able to: Explain the concept of an operon and the function of the operator, repressor, and corepressor Explain the adaptive advantage of grouping bacterial genes into an operon Explain how repressible and inducible operons differ and how those differences reflect differences in the pathways they control

Define control elements and explain how they influence transcription NEED TO KNOW Explain how DNA methylation and histone acetylation affect chromatin structure and the regulation of transcription Define control elements and explain how they influence transcription Explain the role of promoters, enhancers, activators, and repressors in transcription control

Explain how eukaryotic genes can be coordinately expressed NEED TO KNOW Explain how eukaryotic genes can be coordinately expressed Describe the roles played by small RNAs on gene expression Explain why determination precedes differentiation Describe two sources of information that instruct a cell to express genes at the appropriate time

Describe the effects of mutations to the p53 and ras genes NEED TO KNOW Explain how mutations in tumor-suppressor genes can contribute to cancer Describe the effects of mutations to the p53 and ras genes