16 CONTROL OF GENE EXPRESSION

Transcription

1 16 CONTROL OF GENE EXPRESSION Chapter Outline 16.1 REGULATION OF GENE EXPRESSION IN PROKARYOTES The operon is the unit of transcription in prokaryotes The lac operon for lactose metabolism is transcribed when an inducer inactivates a repressor Transcription of the trp operon genes for tryptophan biosynthesis is repressed when tryptophan activates a repressor Transcription of the lac operon is also controlled by a positive regulatory system 16.2 REGULATION OF TRANSCRIPTION IN EUKARYOTES In eukaryotes, of gene expression occurs at several levels Chromatin structure plays an important role in whether a gene is active or inactive Regulation of transcription initiation involves the effects of proteins binding to a gene s promoter and regulatory sites Methylation of DNA can control gene transcription 16.3 POSTTRANSCRIPTIONAL, TRANSLATIONAL, AND POSTTRANSLATIONAL REGULATION Posttransciptional controls mrna availability Translational controls the rate of protein synthesis Posttranslational controls the availability of functional proteins 16.4 THE LOSS OF REGULATORY CONTROLS IN CANCER Most cancers are caused by genes that have lost their normal controls Cancer develops gradually by multiple steps Learning Objectives After reading the chapter, you should be able to: 1. Know the various ways that gene activity (transcription and translation) are turned on (activated) and off (inactivated). 2. Understand how cancer may result when regulatory genes are altered. 3. Be able to list and explain the levels of control in eukaryotes. 4. Understand how prokaryotes control gene expression using operons. 5. Understand the differences in gene expression between prokaryotes and eukaryotes and the fundamental reasons for those differences. 6. Compare mechanisms of the control of gene expression involving RNA processing, translation, and protein structure. Key Terms transcriptional operon promoter transcription unit operator repressor lac operon inducer inducible operon trp operon repressible operon corepressor negative gene positive gene CAP (catabolite activator protein) CAP site Woelker 2009 Control of Gene Expression 135

2 activator coactivators alternative splicing chemotherapy chromatin remodeling promoter proximal region promoter proximal elements enhancer combinatorial gene hormone steroid steroid hormone receptors micro-rnas RNA interference (RNAi) small interfering RNA (sirna) ubiquitin proto-oncogenes oncogenes tumor-suppressor genes multistep progression of cancer general transcription factors transcription initiation complex activators hormone response element DNA methylation silencing genomic imprinting proteosome dedifferentiation benign malignant metastasis Lecture Outline Why It Matters A. A human egg cell remains quiescent until seconds after the cells unite; then it begins a series of divisions that continues as it develops and cells differentiate into distinct types. B. The nucleated cells of the developing embryo retain the same set of genes. The structural and functional differences in cell types are determined not by the presence or absence of certain genes but rather through differences in gene activity. C. The fundamental mechanisms that control gene activity are common to all multicellular eukaryotes. D. The processes that directly control gene activity are collectively called transcriptional, which means the determination of which genes get transcribed into mrna. E. The regulatory mechanisms tailor the production of all cellular molecules. F. The mechanisms of transcriptional and its fine-tuning by additional posttranscriptional, translational, and posttranslational levels are discussed in this chapter Regulation of Gene Expression in Prokaryotes A. Transcription and translation are closely regulated in prokaryotes in ways that reflect the prokaryotic life history. B. Escherichia coli can metabolize a wide range of nutrients, such as milk (lactose), and has a genetic control system that allows it to do so with maximum efficiency. C. The operon is the unit of transcription in prokaryotes. 1. When the environment changes, some metabolic processes are stopped and others are started. Regulation of those genes must be coordinated. The control of these genes is at the transcriptional level. 2. Jacob and Monod (in 1961) proposed the operon model of the control of the expression of genes for lactose metabolism in E. coli. 3. An operon is a cluster of prokaryotic genes and the DNA sequences involved in their. a. One of the DNA sequences is the promoter. b. The cluster of genes transcribed into a single mrna is called a transcriptional unit, which typically catalyzes steps in the same function. 4. The other DNA sequence is the operator short segment to which a regulatory protein binds that can be a repressor (which prevents the operon genes from being expressed) or activator (which turns on expression of the genes). 5. Many operons are controlled by more than one regulatory mechanism, which results in a complex network of superimposed controls that provide total of transcription. D. The lac operon for lactose metabolism is transcribed when an inducer inactivates a repressor. 1. Jacob and Monod researched the genetic controls of lactose metabolisms and showed that three genes are involved: lacz (enzyme that makes β-galactosidase), lacy (enzyme that transports lactose into the cell), and laca (encodes a transacetylace for which the function is unknown). 2. They coined the name operon, the operator for the DNA sequence that controls gene expression. Woelker 2009 Control of Gene Expression 136

3 3. They found that the lac operon was controlled by a regulatory protein termed the lac repressor (Figure 16.2). 4. When lactose is added, the lac operon is turned on, and all three enzymes are synthesized rapidly. Allolactose is an inducer for the lac operon by binding to the lac repressor inactivating it. The lac operon is called an inducible operon. 5. In the absence of lactose, no allolactose inducer molecules inactivate the repressor, and the genes stay turned off. The controls are aided by the fact that bacterial mrnas are very short-lived, about 3 minutes on average. E. Transcription of the trp operon genes for tryptophan biosynthesis is repressed when tryptophan activates a repressor. 1. If tryptophan is absent form the medium, E. coli must make it so that it can synthesize proteins. 2. Tryptophan biosynthesis also involves an operon (Figure 16.4) and is controlled by the trp repressor. The trp repressor is synthesized in an inactive form in which it cannot bind to the operator. 3. When tryptophan is absent from the medium, the trp operon genes are expressed, which is the default state. 4. If tryptophan is present in the medium, there is no need for the cell to make it, and the trp operon is shut off. Tryptophan entering the cell binds to the trp repressor and activates it. 5. This is an example of a repressible operon, and tryptophan acts as a corepressor. 6. Comparing and contrasting the two operons: a. In the lac operon, the repressor is synthesized in active form, the inducer (allolactose) inactivates it, and genes are turned on. b. In the trp operon, the repressor is synthesized in the inactive form, the inducer (tryptophan) activates it, and genes are turned off. 7. Inducible and repressible operons illustrate two types of negative gene. F. Transcription of the lac operon is also controlled by a positive regulatory system. 1. This system ensures that the lac operon is transcribed if lactose is provided as an energy source. 2. The positive gene works when lactose is present and glucose is absent in the growth medium (Figure 16.5). Catabolite activator protein (CAP) is an activator that stimulates gene expression. 3. If both lactose and glucose are present in the medium, the lac operon is not transcribed (Figure 16.5) because glucose is easier to break down and get energy. When glucose is depleted, the bacteria shift to lactose as an energy source. 4. The same positive gene system, using CAP and camp, regulate a large number of other operons. 5. Regulation of gene expression in prokaryotes occurs primarily at the transcriptional level Regulation of Transcription in Eukaryotes A. In eukaryotes, the coordinated synthesis of proteins with related functions also occurs, but the genes are usually scattered around the genomes. B. There are two general categories of eukaryotic gene. 1. Short-term involves gene sets that are quickly turned on or off in response to changes in environmental conditions. 2. Long-term is required for organisms to develop and differentiate. C. In eukaryotes, of gene expression occurs at several levels. 1. The is more complicated because eukaryotes have a nuclear envelope that separates the processes of transcription from translation and gene expression is regulated at more levels than prokaryotes. D. Chromatin structure plays an important role in whether a gene is active or inactive. 1. DNA is wrapped around a core of two molecules each of histones H2A, H2B, H3, and H4, forming a nucleosome (Figure 14.18). 2. Regions of DNA that are tightly wound around histones are inactive. To become active, they must change the state of the chromatin, which is called chromatin remodeling and occurs in two types. a. One, an activator binds to a regulatory sequence upstream of the gene s promoter, and recruits a remodeling complex. b. Two, an activator binds to a regulatory sequence upstream of the gene s promoter, and recruits an enzyme. Woelker 2009 Control of Gene Expression 137

4 E. Regulation of transcription initiation involves the effects of proteins binding to a gene s promoter and regulatory sites. 1. Chromatin remodeling is a critical initial event in gene expression. F. Organization of a eukaryotic protein-coding gene. 1. Immediately upstream of the transcription unit is the promoter (Figure 16.8), often containing the TATA box that when bound then recruits the polymerase. Adjacent to the promoter is the promoter proximal region, which contains promoter proximal elements. G. Activation of Transcription 1. General transcription factors (basal transcription factors) bind to the promoter in the area of the TATA box. When RNA polymerase II binds to this complex, it is now called the transcription initiation complex. 2. Activators are regulatory proteins that control the expression of one or more genes. a. Housekeeping genes are expressed in all cell types and have promoter proximal elements that are recognized by activators present in all cell types. b. The particular set of activators present within a cell at a given time is responsible for determining which genes in that cell are expressed. 3. A coactivator (mediator) is a large multiprotein complex that forms a bridge between the activators at the enhancer, the proteins at the promoter, and the promoter proximal region (Figure 16.10). H. Repression of Transcription 1. The final rate of transcription depends upon the battle between activation signal and the repression signal. 2. Repression in eukaryotes works in various ways. a. Some bind to the same regulatory sequence as activators and block the activators from working. b. Some bind to their own sites nearby and change the activator site so the activator cannot bind. I. Combinatorial Gene Regulation 1. In summary, general transcription factors bind to certain promoter sequences such as the TATA box and recruit RNA polymerase II, and activators bind (promoters/enhancers) and stimulate transcription initiation. 2. Characteristics of any given gene are the number and types of promoter proximal elements. 3. Since some regulatory proteins are activators and others are repressors, the overall effect of regulatory sequences on transcription depends on the particular proteins that bind to them. 4. A relatively small number of regulatory proteins (activators and repressors) control transcription of all protein-coding genes in particular combinations (combinatorial gene ). 5. Because different genes require different combinations of regulatory proteins, the number of genes encoding proteins can be much lower than the number of genes they control. J. Coordinated Regulation of Transcription of Genes with Related Functions 1. There are no operons in eukaryotes, yet transcription of genes with related functions is coordinately controlled. 2. A hormone is a molecule produced by one tissue and transported via the bloodstream to another specific tissue to alter its physiological activity. Testosterone (steroid hormone) regulates the expression of a large number of genes associated with the maintenance of primary and secondary male characteristics. 3. Target tissues have hormone receptors in their cytoplasm that recognize the hormone. 4. All genes regulated by a specific steroid hormone have the same DNA sequence to which the hormone receptor complex binds called the steroid hormone response element. K. Methylation of DNA can control gene transcription. 1. Methylation inhibits transcription and turns genes off, which is called silencing. 2. Genes that code for hemoglobin are highly methylated except in tissues were red blood cells are formed, which have enzymes that remove the methyl groups. 3. DNA methylation in some cases silences large blocks of genes. One of the X chromosomes packs tightly into a mass known as a Barr body in which all genes are turned off. 4. DNA methylation underlies genomic imprinting in which methylation permanently silences transcription of either the inherited maternal or paternal allele of a particular gene. 5. Once mrnas are transcribed from active genes, further occurs at each of the main steps in the pathway from genes to proteins Posttranscriptional, Translational, and Posttranslational Regulation Woelker 2009 Control of Gene Expression 138

5 A. Transcriptional is fine-tuned by these processes. B. Posttranscriptional controls mrna availability. 1. The controls work by several mechanisms including changes in pre-mrna processing and the rate at which mrnas are degraded. C. Variations in Pre-mRNA Processing 1. Pre-mRNAs are processed to produce the finished mrnas, and variations in processing can regulate which proteins are made in cells (see section 15.3 on alternative splicing). Alternative splicing itself is under regulatory control. The outcome is that appropriate proteins within a family are synthesized in which they are optimally functional. D. Posttranscriptional Control by Masking Proteins 1. These controls are important in many animal eggs in which they keep mrnas in an inactive form until the egg has been fertilized. Removal of the masking proteins allows mrna to enter protein synthesis. E. Variations in the Rate of mrna Breakdown 1. The mechanisms involve a regulatory molecule such as a steroid hormone, directly or indirectly affecting the mrna breakdown steps. During milk production, a large amount of casein must be synthesized, and this is accomplished in part by radically decreasing the rate of breakdown of the casein mrna if prolactin is present. 2. Nucleotide sequences in the 5 UTR (untranslated regions; see section 15.3) appear also to be important in determining mrna half life. F. Regulation of Gene Expression by Small RNAs 1. Micro-RNAs (mirnas) regulate important processes, such as development, growth, and behavior. 2. Transcription of the gene produces an RNA that is the precursor to the mirna (Figure 16.13). An enzyme (Dicer) cuts the stem-loop to produce a double-stranded RNA about pairs long, and one degrades, leaving a small single-stranded RNA, the mirna. 3. Researchers think that there are about 120 genes for mirnas in worms and 250 in humans. The mirnas are expressed in developmentally regulated patterns. 4. Silencing a gene posttranscriptionally by a small single-stranded RNA that is complementary to part of an mrna is termed RNA interference (RNAi). The mirnas are one type; another is known as small interfering RNA (sirna), which is not created from nuclear genes. 5. Any gene can be silenced experimentally by RNAi by introducing into the cell a double-stranded RNA that can be processed by Dicer and the protein complex into an sirna complementary to the mrna transcribed from that gene. G. Translational controls the rate of protein synthesis. 1. It occurs in essentially all cells types and species. 2. During early development in most animals, little transcription occurs. The changes in protein synthesis patterns seen in developing cell types and tissues instead derive from the activation, repression, or degradation of maternal mrna. One mechanism for this is the length of the poly (A) tail of the mrna. The length of poly (A) tails is regulated in the cytoplasm, but this process is not completely understood. H. Posttranslational controls the availability of functional proteins. 1. Three main ways this is done are chemical modification, processing, and degradation. a. Chemical modification involves the addition or removal of chemical groups, which reversibly alters the activity of the protein. 2. In processing, proteins are synthesized as inactive precursors, which are converted to an active form under regulatory control. 3. In degradation, typically short-lived proteins are marked for breakdown by enzymes that attach a doom tag, consisting of a small protein called ubiquitin (Figure 16.14). 4. Control of protein breakdown is the last of the opportunities for control of gene expression The Loss of Regulatory Controls in Cancer A. The cell division cycle of all eukaryotic cells is controlled by genes; mutations in these genes can disrupt normal cell growth and division. 1. When dividing and differentiating cells deviate from their normal genetic program, they can give rise to tissue masses called tumors. If the cells stay together, the tumor is benign. 2. If cells in a tumor invade and disrupt surrounding tissue, the tumor is said to be malignant (called cancer). 3. The spreading of a malignant tumor is called metastasis. Woelker 2009 Control of Gene Expression 139

6 B. Most cancers are caused by genes that have lost their normal controls. 1. All characteristics of cancer cells dedifferentiation, uncontrolled division, and metastasis reflect changes in gene activity. 2. Two main types of genes commonly alter activities as cells become cancer cells. a. One class is proto-oncogenes, which encode various kinds of proteins that stimulate cell division. They are altered to become oncogenes by several mechanisms, as follows: (1) mutations in gene s promoter or other control sequences. (2) mutation in the coding segment. (3) translocation, where a segment of chromosome breaks off and attaches to a different chromosome. (4) infecting viruses that may introduce genes to regions in the chromosomes. 3 For example, the activity of MYC, a proto-oncogene controlling cell division, is normally tightly regulated; however, this gene is in an area of the chromosome that often breaks, causing translocation. 4. Several proto-oncogenes encode cell surface receptors that bind extracellular signal molecules, such as peptide hormones or growth factors. 5. Another key group of proto-oncogenes encodes enzyme forming parts of the internal reaction pathways triggered by surface receptors (see Chapter 7). 6. The other main class of genes that shows altered activity in cancer cells is the tumor-suppressor genes, which normally inhibit cell division. C. Cancer develops gradually by multiple steps. 1. In almost all cancers, successive alterations in several to many genes gradually accumulate to tilt normal cells to cancer cells, which is called the multistep progression of cancer (Figure 16.16). 2. The ravages of cancer bring home the critical extent to which humans and all other multicellular organisms depend on the mechanisms controlling gene expression to develop and live normally. Insights from the Molecular Revolution: A Viral Tax on Transcriptional Regulation A. Human T-cell leukemia virus (HTLV) causes a virulent form of cancer, triggering rapid uncontrolled division of white blood cells. The pathway is a G-protein coupled receptor-response pathway involving cyclic AMP. B. In the pathway, a specific activator called CREB (CRE-binding protein) is activated. The binding turns on genes and leads to the rapid division of white blood cells characteristic of the immune response. C. HTLV takes advantage of the pathway by means of a short sequence CGTCA in its DNA that turns on the viral genes and leads to reproduction of the virus; CREB also binds to enhancers of cell division, leading to uncontrolled division of white blood cells, hence leukemia. D. CREB binds only weakly to the viral mimic in a test tube. Wagner and Green found that the HTLV uses one of its own proteins called Tax to get around the problem of weak binding. E. The CREB activator interacts with a DNA as a dimer. Wagner and Green found that the Tax protein greatly increases the ability of CREB to form dimers. F. The Tax protein thus compensates for the imperfection of the viral sequence that imitates CRE by increasing the ability of CREB to form dimers. Woelker 2009 Control of Gene Expression 140