Genetics Padilla: Ch 16 regulation of gene expression in Prokaryotes/Bacteria
Genetics Padilla: Ch 16 regulation of gene expression in Prokaryotes/Bacteria BIOL 3451
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This 14 page Study Guide was uploaded by Jazmine Burnam on Sunday April 3, 2016. The Study Guide belongs to BIOL 3451 at University of North Texas taught by Dr. Padilla in Spring 2016. Since its upload, it has received 189 views. For similar materials see Genetics in Biology at University of North Texas.
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Date Created: 04/03/16
Mechanisms of Gene Regulation- Prokaryotes 1. Define the following a. Gene expression: the process by which the information contained within a gene becomes a useful product Genes can be expressed as either RNA or protein. How is gene expression regulated? Gene regulation in response to normal cellular activity. Gene regulation in response to environmental changes. Videos that might help understanding operons https://www.youtube.com/watch?v=oBwtxdI1zvk b. Polycistronic: Describing a type of messenger RNA that can encode more than one polypeptide separately within the same RNA molecule c. Operon: Way to remember Parts of an operon (PROG) Promoter repressor operator genes Videos that might help understanding operons https://www.youtube.com/watch?v=10YWgqmAEsQ https://www.youtube.com/watch?v=oBwtxdI1zvk https://www.youtube.com/watch?v=oBwtxdI1zvk https://highered.mheducation.com/sites/9834092339/student_vie w0/chapter16/the_lac_operon__induction_.html d. Inducible expression: expression of gene in response to environment. e. Constitutive expression: genes expressed continuously Mechanisms of Gene Regulation- Prokaryotes f. Repressible expression: gene expression inhibited under certain conditions. g. Lactose: a disaccharide sugar derived from galactose and glucose that is found in milk The inducer in gene expression h. DNA regulatory region: Any DNA sequence that is responsible for the regulation of gene expression, such as promoters and operators. A region of a chromosome that controls gene expression. In terms of DNA, it is a regulatory sequence and is the region of the DNA that regulates the transcription of the gene. Mechanisms of Gene Regulation- Prokaryotes 2. Diagram the Lac operon. a. What are the cis-elements and trans-elements involved with regulating the lac operon? Cis-acting elements are DNA sequences that are recognized and bound by the trans-acting elements that regulate transcription. There are two major types of cis-acting elements: promoters and regulatory elements. o Promoters: are the sites where RNA polymerase must bind to the DNA in order to initiate transcription (see "RNA Synthesis and Processing" lecture). The rate or efficiency of promoter use by RNA polymerase is affected by the regulatory elements. o Regulatory Elements are specific DNA sequences that are recognized and bound by the trans-acting elements that stimulate or inhibit the expression of a particular gene. There are two types: enhancers and response elements. Enhancers: are regulatory elements that increase or repress the rate of gene transcription. Response Elements: are regulatory sequences that facilitate the coordinated regulation of a group of genes. Certain ligands such as steroid hormones and cAMP bind to their receptors which in turn bind to their response element to activate or inhibit transcription. Trans-acting factors - factors, usually considered to be proteins that bind to the cis-acting sequences to control gene expression CAP protein b. What is the function for the various components of the lac operon? PROG (promoter-repressor-operator-structural genes) o Promoter: the section of DNA that controls the initiation of RNA transcription o Repressor: any protein that binds to DNA and thus regulates the expression of genes by decreasing the rate of transcription o Operator: a segment of DNA to which a transcription factor protein binds o Structural Genes: are those genes that control the production of a protein that performs a structural role in cell. B-galactosidase Permease Transacetylase c. What are the functions of the proteins coded for by the three structural genes? B-galactosidase: converts lactose to glucose and galactose Permease: facilitate the entry of lactose into the cell Transacetylase: removal of toxic products of lactose digestion d. What protein and molecule regulates this operon in the presence or absence of glucose? This operon is regulated by the Catabolite-Activating Protein (CAP) e. How does the CAP protein impact the function of RNA polymerase? The CAP protein represses the Lac operon If glucose AND lactose present it does not bind CAP facilitates RNA polymerase binding (positive control) 3. Regarding the gene expression regulation of the Lac operon consider the following dietary environments and state whether the structural genes would be expressed. In your answer describe the mechanism that either induces gene expression or represses gene expression in the environments described below. a. lactose only If glucose is absent, then CAP can bind to the operator sequence to activate transcription Lactose binds to the repressor very efficiently and converts the repressor into an inactive state, where it can't bind the Operator. The process is reversed when all the lactose is digested, so the system again will turn off It is induced https://www.tamu.edu/faculty/magill/gene603/PDF%20versions/regulation1.pdf b. lactose and glucose Synthesis of β-galactosidase is not induced until all the glucose has been utilized. The cell will prefer to use glucose More energetically efficient c. glucose only (Constitutive expression): A gene that is not regulated, other than by the strength of its promoter If lactose is absent, then the repressor (not CAP) binds to the operator to prevent transcription. Expression of the lac operon remains low even if lactose is present. Repressor binds to the operator, blocking transcription Nucleotide sequence of operator gene is altered ; no binding occurs; transcription proceeds The repressor protein binds to the operator, preventing transcription from the second promoter. The cAMP concentration is low; as the glucose concentration decreases, the concentration of cAMP increases correspondingly. It is repressed 4. Explain the regulation of the Lac operon if the following mutation is present in E. coli grown either in 1) the presence of lactose and 2) in the absence of lactose: a. mutation in the repressor gene such that the protein cannot bind the operator region cell would continuously produce B galactosidase and the two other enzymes for lactose utilization even in the absence of lactose thus wasting cell resources the structural genes are always turned on Will lead to constitutive expression (no repression in the absence of inducer). b. mutation in the repressor gene such that the protein cannot bind lactose Operon continuously OFF lead to a noninducible phenotype. c. mutation in the operator region such that the repressor protein cannot bind Operator constitutive mutants the binding site for repressor, lead to lower affinity for the repressor and hence less binding. allow continued transcription (and thus expression) of the lac operon even in the absence of inducer; this is referred to constitutive expression. Altered nucleotide sequence in operator region Mutant operator fails to recognize repressor Operon continuously ON d. Mutation in the structural gene such that a premature stop codon is located within the Lac Z gene Altered nucleotide sequence in promoter Mutant promoter fails to bind RNA polymerase Operon continuously OFF 5. How did the analysis of mutations within the Lac Operon contribute to our understanding of gene expression? How has regions of this operon been used in recombinant DNA technology? Research provided insights into the way in which the gene activity is repressed when lactose is absent but induced when it is available The accumulated knowledge in the functioning of the system allowed for its extended use in expression vectors, protein production, and cloning experiments. 6. Why was the analysis of the Lac operon worthy of a Nobel Prize to Lederberg, Jacob and L’Woff? What Genetic laboratory techniques do you think they used to learn about gene regulation? It was very important because back then nothing was known about gene regulation It was the first transcriptionally regulated system to be elucidated. there are many molecular tools and protocols at hand for the high-level production of heterologous proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies You take your gene of interest, clone it in whatever expression vector you have at your disposal, transform it into the host of choice, induce and then, the protein is ready for purification and characterization. 7. Diagram the Trp operon. a. What are the components that regulate the regulation of this operon? Five Structural Genes: o These genes contain the genetic code for the five enzymes in the Trp synthesis pathway One Promoter: o DNA segment where RNA polymerase binds and starts transcription One Operator: o DNA segment found between the promoter and structural genes. It determines if transcription will take place. If the operator is turned "on", transcription will occur. b. What is the function of the protein encoded by the Trp structural genes? the structural genes encode enzymes necessary for the synthesis of typtophan Tryptophan inhibits the first enzyme The more Trp in the cell, the more that can bind to the first enzyme and prevent it from catalyzing the first step. 8. How does the presence or absence of the amino acid Tryptophan play a role in the regulation of the Trp Operon in E. coli? In your answer explain: https://highered.mheducation.com/olcweb/cgi/pluginpop.cgi? it=swf::535::535::/sites/dl/free/0072437316/120080/bio26.swf::The %20Tryptophan%20Repressor a. How Trp amino acid impacts the repressor protein. In absence of trp, the repressor protein dissociates from the operator In presence of trp repressor binds to the operator b. How the presence or absence of Trp impact the structure of the newly transcribed RNA for this operon. If tryptophan is scarce, an mRNA secondary structure referred to as the antiterminator harpin is formed (bottom right).. Transcription proceeds past the antiterminator harpin region, and the entire mRNA is subsequently produced In the presence of excess tryptophan, the mRNA structure that is formed is referred to as a terminator hairpin (upper left), and transcription is almost always terminated prematurely, just beyond the attenuator. c. How the leader region sequence, after it is transcribed, impacts the expression of this operon. When trp is present and the trp operon is repressed, initiation of transcription still occurs at a low level, but is subsequently terminated, instead continuing beyond the leader sequence into the structural genes and It undergoes attenuation (weakens/impairs) expression of the operon When trp is absent, or present in low concentrations, transcription is initiated but is not subsequently terminated, instead continuing beyond the leader sequence into the structural genes d. How the amount of Trp influences the expression of this operon? , The more Trp in the cell, the more that can bind to the first enzyme and prevent it from catalyzing the first step.
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