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The First Page of Teaching Plan No. course Biochemistry specialty clinic class 2015-2 teacher Chen yan period 3h students’ level undergraduate professional title Biochemistry associate professor time of writing 2016.12 chapter Regulation of gene expression time of using 2016-2017(1) objectives and requirements keys and difficulties updated information arrangement teaching methods books and references teachers’ group discussion about the plan 1.Master the concept of regulation of gene expression and the regulation of transcription in prokaryotes. 2.Familiar with the characteristics of regulation of transcription in prokaryotes, the structure of genome in eukaryotes, the specificity and features of gene expression. 3.Understand the regulation of transcription gene expression in eukaryotes at the level of transcription. Keys: Concept of gene expression regulation; Prokaryotic gene expression regulation Difficulties: lac operon and its mechanism in prokaryotes; cis-acting element and trans-acting factor no review the content last class(5min); the definition of gene regulation (10min); Principles of Gene Regulation(30min); operon (15min); lac operon and its mechanism (50min); cis-acting element and trans-acting factor (35min); discuss and summarize(5min). Using CAI to explain, enlightening method Lippincott’s illustrated review :Biochemistry Pamela C. Champe wilkins 2009 Biochemistry the second edition High Education Press 2002 author: Reginald H. Garrett, Charles M. Grisham According to learn the principles of gene regulation, then to understand prokaryotic and eukaryotic gene expression. The lac operon and its mechanism in prokaryotes should be lectured clearly. Agree apply in class. comments from the department Lippincott’s willam & Sign name: (Content) Lesson plan for page Chapter 12 Regulation of Gene Expression I.Teaching Goals It is based on a mastery of basic concepts and principles of regulation and control of gene expression, then study the regulation of transcription in prokaryotes and to know(understand) the regulation of transcription gene expression in eukaryotes at the level of transcription. II.Teaching Demands 1.Master the concept of regulation of gene expression and the regulation of transcription in prokaryotes. 2.Familiar with the characteristics of regulation of transcription in prokaryotes, the structure of genome in eukaryotes, the specificity and features of gene expression. 3.Understand the regulation of transcription gene expression in eukaryotes at the level of transcription. III.Teaching Contents 1. The basic concepts and principles of regulation and control of gene expression The concept of gene expression, temporal and spatial specificity and the features of gene expression it includes constitutive gene expression, induction and repression. 2. The basic principle in regulation of gene expression The regulation of gene expression in multi-levels and basic factor of gene transcription activation. 3. The regulation of gene expression in prokaryotes The feature in regulation of prokaryotic gene transcription. The mechanism of gene transcription at initiation regulated by lac operon in E.coil. 4. The regulation of gene expression in eukaryotes The feature of eukaryotic genome structure, the concept of cis-acting element and trans-acting factor. Study the feature of eukaryotic gene expression and transcription regulation of RNA polⅡat initiation by self. IV. Teaching period 3h Lesson plan for page (Content) Chapter 12 Regulation of Gene Expression Organisms adapt to environmental changes by altering gene expression. In order for the organism to adapt to its environment and to conserve energy and nutrients, the expression of genetic information must be cued to extrinsic signals and respond only when necessary. Mammalian cells possess about 1000 times more genetic information than does the bacterium Escherichia coli. Much of this additional genetic information is probably involved in regulation of gene expression during the differentiation of tissues and biologic processes in the multicellular organism and in ensuring that the organism can respond to complex environmental challenges. Control of transcription ultimately results from changes in the interaction of specific binding regulatory proteins with various regions of DNA in the controlled gene. This can have a positive or negative effect on transcription. Transcription control can result in tissue-specific gene expression, and gene regulation is influenced by hormones, heavy metals, and chemicals. In addition to transcription level controls, gene expression can also be modulated by gene amplification, gene rearrangement, posttranscriptional modifications, and RNA stabilization. Analysis of the regulation of gene expression in prokaryotic cells helped establish the principle that information flows from the gene to a messenger RNA to a specific protein molecule. These studies were aided by the advanced genetic analyses that could be performed in prokaryotic and lower eukaryotic organisms. In this chapter, the initial discussion will center on prokaryotic systems. 1.the definition of gene regulation Regulation of gene expression (gene regulation) refers to the cellular control of the amount and timing of changes to the appearance of the functional product of a gene. Although a functional gene product may be an RNA or a protein, the majority of the known mechanisms regulate the expression of protein coding genes. Any step of the gene's expression may be modulated, from DNA-RNA transcription to the post-translational modification of a protein. Gene regulation gives the cell control over its structure and function, and is the basis for cellular differentiation, morphogenesis and the versatility and adaptability of any organism. Lesson plan for page (Content) 2.Principles of Gene Regulation 2.1 the definition of gene expression Genes are functional units of DNA that contain the instructions for making proteins or RNA. And it is the basic unit in inheritance. A genome is the complete collection of hereditary information for an individual organism. Homo sapiens, for example, have 22 autosomes plus an X chromosome or Y chromosome. Gene expression is the process by which a gene's DNA sequence is converted into the functional proteins of the cell. 2.2 The specificity of gene expression 2.2.1 Temporal specificity Requirements for some gene products change over time. The need for enzymes in certain metabolic pathways may wax and wane as food sources change or are depleted. This protein is alpha-fetoprotein, a major plasma protein produced by the yolk sac and the liver during fetal life. Expression of AFP (Alpha-fetoglobulin) in adult cells is low, however synthesis aberrantly occurs in adult liver cancer cells. Alpha-fetoprotein expression in adults is often associated with hepatoma or teratoma. However, hereditary persistance of alpha-fetoprotein may also be found in individuals with no obvious pathology. The protein is thought to be the fetal counterpart of serum albumin, and the alpha-fetoprotein and albumin genes are present in tandem in the same transcriptional orientation on chromosome 4. 2.2.2 Spatial specificity (Tissue-specific regulation) Specialization of cellular function can dramatically affect the need for various gene products; an example is the uniquely high concentration of a single protein—hemoglobin—in erythrocytes. Given the high cost of protein synthesis, regulation of gene expression is essential to making optimal use of available energy. Adult tissue-specific stem cells have the capacity to self-renew and generate functional differentiated cells that replenish lost cells throughout an organism’s lifetime. Lesson plan for page (Content) 2.3 Features of Gene Regulation 2.3.1 housekeeping gene The expression of some genes is constitutive, essential for basic processes involving in cell replication and growth. It means that they are expressed at a reasonably constant rate and not known to be subject to regulation. Genes for products that are required at all times, such as those for the enzymes of central metabolic pathways, are expressed at a more or less constant level in virtually every cell of a species or organism. These are often referred to as housekeeping genes. Unvarying expression of a gene is called constitutive gene expression. 2.3.2 inducible gene An inducible gene is one whose expression increases in response to an inducer or activator, a specific positive regulatory signal. In general, inducible genes have relatively low basal rates of transcription. By contrast, genes with high basal rates of transcription are often subject to down-regulation by repressors. 2.4 Features of Regulation Inducer - The substance which induces gene expression or protein synthesis is called as inducer. The phenomenon is called as induction. Repressor - The substance which stops or represses the expression of specific genes is called as repressor and the phenomenon is called as repression. If the presence of a specific regulatory element enhances the level of gene expression, then that is called as positive regulation and the molecule is called as activator, e.g. lac operon If the presence of a specific regulatory molecule will reduce the gene expression, then it is called as negative regulation and the molecule is called as repressor. Jacob and Monad in 1961 proposed a model which explains how a bacteria metabolizes lactose and tryptone, and the model is called as Jacob-Monad model. Bacteria use a positive control to metabolize lactose and negative control or regulation to metabolize tryptone. Lesson plan for page (Content) 2.5. Regulated stages of gene expression(eurkaryote) Any step of gene expression may be modulated, from the DNA-RNA transcription step to post-translational modification of a protein. Following is a list of stages where gene expression is regulated: Chemical and structural modification of DNA or chromatin; Transcription; Translation; Post-transcriptional modification; RNA transport; mRNA degradation; Post-translational modifications; 3.Gene Control in Prokaryotes In bacteria, genes are clustered into operons: gene clusters that encode the proteins necessary to perform coordinated function, such as biosynthesis of a given amino acid. RNA that is transcribed from prokaryotic operons is polycistronic a term implying that multiple proteins are encoded in a single transcript. --control of gene expression enablesindividual bacteria to adjust theirmetabolism to environmental change --cells vary amount of specific enzymesby regulating gene transcription -turn gene on or turn gene off ex. if you have enough tryptophanin your cell then you don’t need to make enzymesused to build tryptophan -waste of energy -turn off gene which codes for enzyme The activity of RNA polymerase at a given promoter is in turn regulated by interaction with accessory proteins, which affect its ability to recognize start sites. These regulatory proteins can act both positively (activators) and negatively (repressors). The accessibility of promoter regions of prokaryotic DNA is in many cases regulated by the interaction of proteins with sequences termed operators. The operator region is adjacent to the promoter elements in most operons and in most Lesson plan for page (Content) cases the sequences of the operator bind a repressor protein. However, there are several operons in E. coli that contain overlapping sequence elements, one that binds a repressor and one that binds an activator. As indicated above, prokaryotic genes that encode the proteins necessary to perform coordinated function are clustered into operons. Two major modes of transcriptional regulation function in bacteria (E. coli) to control the expression of operons. Both mechanisms involve repressor proteins. One mode of regulation is exerted upon operons that produce gene products necessary for the utilization of energy; these are catabolite-regulated operons. The other mode regulates operons that produce gene products necessary for the synthesis of small biomolecules such as amino acids. Expression from the latter class of operons is attenuated by sequences within the transcribed RNA. A classic example of a catabolite-regulated operon is the lac operon, responsible for obtaining energy from β-galactosides such as lactose. A classic example of an attenuated operon is the trp operon, responsible for the biosynthesis of tryptophan. Operon - An operon is a set of genes which are linked and are under the control of one promoter or operator. These genes accomplish one single task. An operon basically consists of two categories of genes. 1.Structural genes - These genes are segments of DNA, which code for functional peptides, or enzymes or proteins. Proteins of structural genes directly interact with the inducer or accomplish a single task. 2.Control genes - These are genes which are primarily responsible for controlling the structural genes by producing an inducer or repressor substance. There are basically three types of genes. i. Regulator genes - The regulator gene (R) produces some specific enzymes which act as repressor substances. This repressor binds to the operator gene and thus stops the expression of structural genes. ii. Promoter gene - The promoter gene (P) is the DNA segment at which RNA polymerase binds. It initiates the transcription of the structural genes. iii. Operator gene -The operator gene (O) is the segment of DNA which exercises a control over transcription. It lies close to the structural gene and the repressor binds to it. Lesson plan for page (Content) Lactose Operon in E coli –A inducible operon In an inducible operon, the repressor actively blocks the gene from transcription. The controller molecule attaches to the repressor removing it from the operator and transcription proceeds. 1.Structure and catabolite of glucose The molecular mechanisms responsible for the regulation of the genes involved in the metabolism of lactose are now among the best-understood in any organism. β-Galactosidase hydrolyzes the β-galactoside lactose to galactose and glucose. The structural gene for β-galactosidase (lacZ) is clustered with the genes responsible for the permeation of galactose into the cell (lacY) and for thiogalactoside transacetylase (lacA). The structural genes for these three enzymes, along with the lac promoter and lac operator (a regulatory region), are physically associated to constitute the lac operon. This genetic arrangement of the structural genes and their regulatory genes allows for coordinate expression of the three enzymes concerned with lactose metabolism. When E coli is presented with lactose or some specific lactose analogs under appropriate nonrepressing conditions (eg, high concentrations of lactose, no or very low glucose in media; see below), the expression of the activities of β-galactosidase, galactoside permease, and thiogalactoside transacetylase is increased 100-fold to 1000-fold. Upon removal of the signal, ie, the inducer, the synthesis of these three enzymes declines. When E coli is exposed to both lactose and glucose as sources of carbon, the organisms first metabolize the glucose and then temporarily stop growing until the genes of the lac become induced to provide the ability to metabolize lactose as a usable energy source. Although lactose is present from the beginning of the bacterial growth phase, the cell does not induce those enzymes necessary for catabolism of lactose until the glucose has been exhausted. This phenomenon was first thought to be attributable to repression of the lac operon by some catabolite of glucose; hence, it was termed catabolite repression. It is now known that catabolite repression is in fact mediated by a catabolite gene activator protein (CAP) in conjunction with cAMP. This protein is also referred to as the cAMP regulatory protein (CRP). The expression of many inducible enzyme systems or operons in E coli and other prokaryotes is sensitive to catabolite repression, as discussed below. Lesson plan for page (Content) 2. regulation (1)negative regulation The physiology of induction of the lac operon is well understood at the molecular level.Expression of the normal lacI gene of the lac operon is constitutive; it is expressed at a constant rate, resulting in formation of the subunits of the lac repressor. Four identical subunits with molecular weights of 38,000 assemble into a lac repressor molecule. The LacI repressor protein molecule, the product of lacI, has a high affinity for the operator locus. The operator locus is between the promoter site, at which the DNA-dependent RNA polymerase attaches to commence transcription, and the transcription initiation site of the lacZ gene. When attached to the operator locus, the LacI repressor molecule prevents transcription of the operator locus as well as of the distal structural genes, lacZ, lacY, and lacA. Thus, the LacI repressor molecule is a negative regulator; in its presence (and in the absence of inducer), expression from the lacZ, lacY, and lacA genes is prevented. A lactose analog that is capable of inducing the lac operon while not itself serving as a substrate for β-galactosidase is an example of a gratuitous inducer. An example is isopropylthiogalactoside (IPTG). The addition of lactose or of a gratuitous inducer such as IPTG to bacteria growing on a poorly utilized carbon source(such as succinate) results in prompt induction of the lac operon enzymes. Small amounts of the gratuitous inducer or of lactose are able to enter the cell even in the absence of permease. The LacI repressor molecules—both those attached to the operator loci and those free in the cytosol—have a high affinity for the inducer. Binding of the inducer to a repressor molecule attached to the operator locus induces a conformational change in the structure of the repressor and causes it to dissociate from the DNA because its affinity for the operator is now 103 times lower than that of LacI in the absence of IPTG. If DNA-dependent RNA polymerase has already attached to the coding strand at the promoter site, transcription will begin. Derepression of the lac operon allows the cell to synthesize the enzymes necessary to catabolize lactose as an energy source. Based on the physiology just described, IPTG-induced expression of transfected plasmids bearing the lac operator-promoter ligated to appropriate bioengineered constructs is commonly used to express mammalian recombinant proteins in E coli. Lesson plan for page (Content) (2)positive regulation In order for the RNA polymerase to efficiently form a PIC at the promoter site, there must also be present the catabolite gene activator protein (CAP) to which cAMP is bound. By an independent mechanism, the bacterium accumulates cAMP only when it is starved for a source of carbon. In the presence of glucose—or of glycerol in concentrations sufficient for growth—the bacteria will lack sufficient cAMP to bind to CAP because the glucose inhibits adenylyl cyclase, the enzyme that converts ATP to cAMP . Thus, in the presence of glucose or glycerol, cAMP-saturated CAP is lacking, so that the DNA-dependent RNA polymerase cannot initiate transcription of the lac operon. In the presence of the CAP-cAMP complex, which binds to DNA just upstream of the promoter site, transcription then occurs, and stimulates RNA transcription 50-fold. Studies indicate that a region of CAP contacts the RNA polymerase α subunit and facilitates binding of this enzyme to the promoter. Thus, the CAP-cAMP regulator is acting as a positive regulator because its presence is required for gene expression. The lac operon is therefore controlled by two distinct, ligand-modulated DNA binding trans factors; one that acts positively (cAMP-CRP complex) and one that acts negatively (LacI repressor). Maximal activity of the lac operon occurs when glucose levels are low (high cAMP with CAP activation) and lactose is present (LacI is prevented from binding to the operator).When the lacI gene has been mutated so that its product, LacI, is not capable of binding to operator DNA, the organism will exhibit constitutive expression of the lac operon. In a contrary manner, an organism with a lacI gene mutation that produces a LacI protein which prevents the binding of an inducer to the repressor will remain repressed even in the presence of the inducer molecule, because the inducer cannot bind to the repressor on the operator locus in order to derepress the operon. Similarly, bacteria harboring mutations in their lac operator locus such that the operator sequence will not bind a normal repressor molecule constitutively express the lac operon genes. Mechanisms of positive and negative regulation comparable to those described here for the lac system have been observed in eukaryotic cells. Lesson plan for page (Content) 4.Gene expression in eukaryotic cells --the control mechanisms of gene expression are varied and complex --cellular differentiation of eukaryotic cells depends on turning on and off genes in the proper sequence (1)DNA packing in eukaryotic chromosomes helps regulate gene expression --DNA wrapping around histonesand other proteins into nucleosomes, coiling, supercoiling, and additional folding into chromosomes --DNA packing prevents gene expression, most likely by preventing transcription (2)Initiation of transcription -Complex assemblies of proteins control eukaryotic transcription --Transcription factors interact with enhancer sites (a DNA sequence) in regulating the binding of RNA polymerase to a gene’s promoter --The binding of activators to enhancers initiates transcription --Repressor proteins interact with nucleotide sequences called silencers -inhibit the start of transcription Like the prokaryotic gene, the eukaryotic gene has a number of regions, each important to transcription. The components of the eukaryotic transcription complex include: CIS Acting Elements (Control elements) A specific promoter region within the control elements, which indicates the starting point for transcription. Eukaryotic genes are regulated by promoter elements located just upstream (5 ') from the transcription initiation sites in a manner quite similar to the regulation of prokaryotic genes. In addition to the nearby promoters, many eukaryotic genes are also regulated by more distant cis acting elements called enhancers and silencers. Lesson plan for page (Content) A region called the enhancer that stimulates the binding of RNA polymerase to the promoter region. The enhancer region is comprised of non-coding DNA that binds to transcription factors called activators. Activators fold the DNA so that the enhancers are brought to the promoter region of the gene where they bind to additional transcription factors. Silencers are control elements that can inhibit transcription. A transcription factor that binds to a silencer control element and blocks transcription is called a repressor. Specific Transcription Factors Eukaryotic transcription begins with the formation of a pre initiation complex formed by the amalgamation of a group of general transcription factors. Proteins that exert control over transcription at specific promoters are the specific transcription factors. These proteins generally have two domains, a domain that recognizes a specific DNA sequence and a domain that recognizes another protein like the pre initiation complex. A majority of specific transcription factors act by recruiting the components of the RNA polymerase holoenzyme. (3)Eukaryotic RNA may be spliced in more than one way Introns have been shown to function in gene regulation --alternative RNA splicing --increasing the possibility of crossovers between exons --increasing genetic diversity (4)Regulation in the cytoplasm mRNA breakdown: long-lived mRNAs get translated into many protein molecules than do short-lived ones translation Protein alterations: Post-translational control mechanisms in eukaryotes often involve cutting polypeptides into smaller, active final products Protein breakdown