BIOCHEMISTRY REGULATION OF GENE EXPRESSION & MUTATIONS PDF

BIOCHEMISTRY REGULATION OF GENE EXPRESSION & MUTATIONS Sheila Marie P. Torres Trans Group: 1E, 2E Polypeptide for...

BIOCHEMISTRY REGULATION OF GENE EXPRESSION & MUTATIONS Sheila Marie P. Torres Trans Group: 1E, 2E Polypeptide forms a structural protein or an enzyme OUTLINE These functional proteins confer a specific trait to I. Learning Objectives an organism or are essential to cell metabolism II. Types of Gene Regulation and function III. Regulation of Gene Expression Some proteins may also be involved in the control IV. Control of Transcription of gene expression A. Types of Genes Read Harper’s Illustrated Biochemistry, 30th Edition, B. Five Important Components of a Gene Chapter 38, pg. 429 and Prerequisite Reading, pg. 2 for V. Regulators further information. VI. Regulation of Gene Expression in Prokaryotes A. Operon 1. Lac Operon 2. Tryptophan Operon VII. Regulation by Attenuation A. Attenuation in Histidine Operon VIII. Mechanism of Regulation of Gene Expression In Eukaryotes A. Chromatin Remodeling/Nucleosome Remodelling B. Covalent Modification of the Histone Tails 1. Epigenetics Effect on Gene Regulation Figure 1. Regulation of Gene Expression C. DNA Methylation D. Gene Loss CONTROL OF TRANSCRIPTION E. Gene Amplification Regulation of gene expression is an essential process in IX. Transcriptional Level of Control maintaining the integrity of a cell. X. Hormones Prokaryotic cell - use operons in regulating expression of A. Control of Gluconeogenesis by Response Elements the genes. XI. Mutations Eukaryotic cell - (due to complexity), use a variety of mechanisms in order to decrease or increase the expression REFERENCES: of a gene Dr. Sheila Marie P. Torres— Prerequisite Reading, PPT The most common form of regulating gene expression is: & Recorded Lecture “regulation by actually changing the rate of transcription. Transcription can be downregulated or upregulated by NUMBER OF PAGES: 00 number of mechanisms LEARNING OBJECTIVES A. TYPE OF GENES At the end of this video lecture, the student must be able to: Inducible: needs an inducer/activator Accurately discuss control of gene regulation at the Constitutive: housekeeping genes, genes that are transcriptional level expressed at a constant rate. Discuss correctly how response elements affect metabolism Source: Prerequisite Reading, pp. 2 Accurately relate the different types of genetic mutations to a particular genetic disease B. FIVE IMPORTANT COMPONENTS OF GENE TYPES OF GENE REGULATION 1. Positive Regulation Expression of the genetic information is quantitatively increased by the presence of a specific regulatory element (activator/inducer) 2. Negative Regulation Expression of the genetic information is diminished by specific regulatory elements (repressor) REGULATION OF GENE EXPRESSION Figure 2. Five components of a Gene Regulation of Gene Expression, Gene Regulation and Exons Mutation Timestamp: 1:27 to 2:24 ○ Important segments of the eukaryotic gene that code for a Occurs at different steps of gene expression polypeptide. They are usually separated by intervening ○ But gene expression is controlled mainly at transcription sequences known as introns. level Introns DNA forms an RNA molecule such as mRNA, tRNA, and ○ Do not code for useful polypeptides. These are sections rRNA. that are removed during RNA processing particularly ○ These RNA molecules are needed for the formation of a during RNA splicing bringing exons together. polypeptide chain. Transcription Start Site Regulation of Gene Torres 1 Expression and Mutation  ○ Initiation of transcription takes place on this site. This is NF-1 CCAATT box where RNA polymerase binds and begins the process of transcription. Steroid Receptor HRE Promoter ○ Core Promoter cAMP response element CRE response element (CREB) protein Contains a consensus sequence, which means that the sequence will remain consistent or the same in all Table 1. Other examples and cis-acting and trans-acting chains of a eukaryotic organism. Therefore, it can be elements. Cis-acting are the specific sequence of observed in all cell types and in all tissue types nucleotides, while trans-acting elements are the general Ex: TATA box (has the consensus sequence TATA transcription factors that bind to these sequences. in all cell type) Function: It is the binding site for all essential REGULATION OF GENE EXPRESSION IN PROKARYOTES transcription factors and regulatory proteins, forming a large transcription factor-protein complex. This OPERON complex is essential for transcription to take place. a cluster of genes that are transcribed together under a The formation of this complex on the TATA box single promoter, which controls the expression of these recruits the binding of the other proteins. This large related genes. complex now interacts with the proteins that bind to the transcription start site, and begin transcription. TYPES OF AN OPERON Example: transcription factor IID contains the TATA Lac Operon binding protein that recognizes and binds to the TATA box. Transcription factor IIB, on the other hand, assists TATA binding protein to interact with RNA polymerase. Upstream Promoter ○ Allows the binding of the other proteins that can either activate or inhibit transcription. The number, type, and sequence of the upstream promoter vary from organism to organism. a) Example: CCAAT and the GC rich region that binds general transcription factors. 2) Enhancer (Response Element) a) May be up to 1000 bp away from the gene. Figure 3. Lac Operon ‘on’, repressor inactive, lactose They may be located upstream, downstream, present (see: Prerequisite Reading) or within an intron of the gene. Enhancer can bind a special transcription factor and once ○ Consists of: this transcription factor binds to the enhancer, Regulatory gene (lac i): produces the repressor it loops or it bends around the DNA like so, protein putting a transcription factor protein complex Promoter site (P): where RNA polymerase binds to on the promoter and then stimulating start transcription transcription–This is just one mechanism that Operator site (O): where lac repressor binds to block a eukaryotic organism regulates their way of RNA polymerase from binding to promoter site and transcription. thus prevent transcription Three (3) Structural genes: REGULATORS lacZ (for β-galactosidase) - hydrolyzes lactose into Silencers - these are specific sequences of nucleotides glucose and galactose found in the gene. lacY (for permease) - transport of lactose across ○ Binding site for transcription factors and regulatory the cell membrane proteins lacA (for thiogalactoside transacetylase) ○ May be an activator or repressor ○ Turned off by default but inducible upon presence of Activator Proteins - bind to the enhancers lactose. Repressor Proteins - bind to silencers ○ Regulation of Lac Operon Categorized based on location uses lactose as energy source upon absence of ○ Cis-acting regulators - DNA sequences to which glucose transcriptions factors and activators bind bacteria usually depends on glucose as primary Exert only on nearby genes such as TATA Box, energy source Enhancers, and Core Promoter regions negative regulation by lac repressor in the ○ Trans-acting regulators - include all regulatory protein absence of lactose and presence of Glucose that bind to Cis acting regulators Lac repressor binds to the O site → blocks RNA General transcription factors, specific transcription Pol access of the structural genes factors, activators, and repressors No transcription and translation Gene that produces general transcription factors positive control by induction of expression in the are under trans-acting regulators presence of lactose and absence of Glucose Permease allows transport of lactose through the Transcription Factor (DNA Response Element cell membrane Binding Protein) (Binding Site) β-galactosidase cleaves lactose into glucose and SP-1 GC-rich galactose allolactose is formed by combining glucose and galactose through α1-6 linkage Regulation of Gene Torres 2 Expression and Mutations  BIOCHEMISTRY REGULATION OF GENE EXPRESSION & MUTATIONS Sheila Marie P. Torres Trans Group: 1E, 2E allolactose serves as inducer and binds the His Operon contains genes for histidine synthesis and repressor protein produces histidine enzymes for the biosynthetic pathway of repressor protein cannot bind the O site due to histidine when histidine is not available in the medium. conformational change in its structure ○ When histidine is available, the operon is turned off. RNA Pol is allowed to have basal transcription of ○ Attenuation is one way to control His Operon. the structural genes Transcription is constitutively initiated at the 5’ UTR of Enhance by CAP-binding protein mRNA. positive control by catabolite repression in the As soon as the Shine Dalgarno appears at the 5’ UTR presence of glucose and lactose of the mRNA, ribosome binds to translate the 5’ UTR Glucose is metabolized first (leader sequence) into a non functional protein known Glucose inhibits the high rate of transcription by as the leader peptide. lowering the concentration of cAMP If histidine is abundant, the ribosome easily finds When glucose is exhausted, cAMP concentration His tRNA and the leader peptide is formed. increases A secondary structure, hairpin loop, is formed. cAMP binds to CAP-binding protein RNA Pol stops transcription before it reaches the the complex binds to the CAP-binding site in the structural genes. promoter In the absence of histidine, the ribosome stalls at this enhances transcription of the structural genes histidine codon because it cannot easily find His by the RNA polymerase tRNA. Source: Prerequisite Reading pp. 2-4 The message does not fold into a secondary Tryptophan Operon structure. ○ Genes for the enzymes of tryptophan biosynthetic RNA Pol continues to transcribe the structural pathway genes. ○ Turned off by the presence of a repressor (repressible gene) MECHANISM OF REGULATION OF GENE EXPRESSION IN EUKARYOTES 1. Chromatin Remodeling/Nucleosome Remodeling 2. Covalent Modification of the Histone Tails through Acetylation 3. DNA Methylation 4. Gene Amplification 5. Gene Deletions DNA of eukaryotic organisms are not naked and are complex with histones. The nucleosome core provides a barrier for the formation of the transcriptional apparatus especially Figure 4. Tryptophan Operon when the promoter is included in the nucleosome core. ○ Apo-repressor (inactive repressor) is Hence, the need for chromosome remodeling. produced by the regulatory gene Source: Prerequisite Reading, pp. 5-7 ○ Proteins necessary for tryptophan synthesis Read Harper’s Illustrated Biochemistry, 32nd Edition, are produced by the 5 structural genes of the Chapter 38, pp. 429-437 Tryptophan operon ○ The Tryptophan operon is turned on during CHROMATIN REMODELING/NUCLEOSOME REMODELING the absence of Tryptophan and the Apo-repressor (inactive repressor) cannot bind the operator site ○ Transcription is repressed when there is an excess in Tryptophan ○ Tryptophan acts as a co-repressor, activating the Apo-repressor (inactive repressor) ○ Binding of the activated repressor blocks RNA Pol transcription of the structural genes Source: Prerequisite Reading pp. 4-5 REGULATION BY ATTENUATION Source: Prerequisite Reading p. 5 ○ Regulation by premature termination of transcription. ○ Depends on coupled transcription and translation. Figure 5. Chromatin Remodeling (Prerequisite reading) ○ Never occur in eukaryotes. Displacement of the nucleosome from specific DNA ATTENUATION IN THE HISTIDINE OPERON sequences. Regulation of Gene Torres 3 Expression and Mutation  ATP driven. Activates the gene. COVALENT MODIFICATION OF THE HISTONE TAILS Source: Prerequisite Reading (page 6) Through acetylation ○ Histone acetyltransferases (HATs) transfer the acetyl group from acetyl coA to lysine residues in the tail of the histone octamer. ○ Reduces electrostatic interaction between histone and the DNA. Unwinding of the DNA from histones. ○ Some repressor or co-repressor contains Histone deacetylases that remove acetyl groups and inactivate the gene. EPIGENETICS EFFECT ON GENE REGULATION Figure 6. Transcriptional Level. (Prerequisite reading) Source: Prerequisite Reading (page 6) Chemical modifications of DNA Enhancers and silencers are DNA sequences that can ○ There is no change in the base sequence and therefore control activities of the gene although they are hundreds of not a mutation. base pairs away from the promoter ○ It usually involves methylation of cytosine in CG Activators and Repressor proteins bind to these elements. sequences. These proteins are known as special transcription factors. ○ Such methylation usually silences the gene. Example: extreme condensation silences the gene TRANSCRIPTION FACTORS like what happened in heterochromatin. Activator proteins that bind response elements. Heterochromatin is highly compacted during With 2 recognizable domains: interphase and is found in regions near the ○ DNA-binding Domain centromere. Binds to specific nucleotide sequence in the promoter Constitutive heterochromatin remains condensed or response element. most of the time in all cells (Euchromatin on the Used to define certain families of transcription factors. other hand contains all active genes that are ○ Activation domain always transcribed). Binds to other transcription factors. Methylation on the CPG island of the FMR1 gene Interact to RNA Pol II to stabilize the formation of the silences the gene leading to none production of initiation complex. the FMR1 protein in Fragile x Syndrome Recruit chromatin modifying proteins such as HATs Heterochromatin. (histone acetyltransferases) or HDATs. DNA METHYLATION SELECTED DNA BINDING MOTIFS Cytosine on both strands may be methylated to Helix-turn-helix 5-methylcytosine (Inactivates the gene). ○ Homeodomain Maintenance of inactive heterochromatin. Zinc fingers (steroid hormone receptor) Most important type of gene regulation in preventing the ○ Cys4 zinc finger transcription of the genes intended to be permanently turned ○ Cys2 His2 zinc finger (e.g. TFIIIA) off. Basic domains Occurs in the CpG islands (usually near promoters) ○ Leucine zippers factors (bZIP) Housekeeping genes - CpG islands are unmethylated ○ Basic helix-loop-helix (bHLH) Tissue specific genes - CpG islands may be methylated to Beta-scaffold factors with minor groove contacts silenced the genes ○ HMG (High mobility group) proteins GENE LOSS Transcription Response Function Protein Genes are deleted or partially deleted from cells factor element class Non production of functional protein (DNA-binding (binding protein) site) GENE AMPLIFICATION SP-1 GC-rich basal Zinc finger Certain regions of chromosomes undergo repeated cycles of replication NF-1 CCAAT basal - box Not a usual physiological means The newly synthesized DNA is excised and form small Steroid HRE steroid Zinc finger unstable chromosomes called double minutes receptor response ○ Integrate into other chromosome throughout the genome therefore amplifying the gene cAMP CRE response to Leucine response response cAMP zipper element element TRANSCRIPTIONAL LEVEL OF CONTROL (CREB) protein Source: Pre-requisite readings (pp. 7-8) Homeodomain - Regulation Helix turn proteins during helix development Regulation of Gene Torres 4 Expression and Mutations  BIOCHEMISTRY REGULATION OF GENE EXPRESSION & MUTATIONS Sheila Marie P. Torres Trans Group: 1E, 2E Table 2. Properties of some common transcription factors 3. Protein kinase A becomes active. (Source: Pre-requisite reading, p. 8) 4. Protein kinase A phosphorylated CREB and activates it. SP-1 and NF-1 are general transcription factors whereas the 5. Activated CREB enters the nucleus and binds to the CRE rest are specific transcription factors. associated with the PEPCK gene → Increases gene TFIID (TATA factor) is also a kind of general transcription expression factor that binds the TATA box. 6. Increases PEPCK in the cell → Increases rate of gluconeogenesis HORMONES Module 1B #13, Gene Regulation and Mutation Video Effect of cortisol on PEPCK gene Lecture, 9:50-13:58 The expression of the gene is also increased or stimulated by regulate the activity of some transcription factors. (ex. steroid the following sequence: receptors and the CREB protein) 1. Cortisol diffuses into the hepatocyte (where it binds to the ○ E.g. control of gluconeogenesis by response elements. receptor) 2. The complex enters the nucleus and binds to the CONTROL OF GLUCONEOGENESIS BY RESPONSE Glucocorticoid Response Element (GRE), which is also ELEMENTS associated with PEPCK gene 3. Binding of the cortisol and its receptor to the GRE results to an increase in the gene expression 4. Once the gene expression is stimulated, the concentration of the PEPCK enzyme also increases in the cell and, therefore, Increases rate of gluconeogenesis *CREB= cAMP Response Element-Binding protein *PEPCK= ​Phosphoenolpyruvate carboxykinase *GRE= Glucocorticoid Response Elements *cAMP= Cyclic adenosine monophosphate Figure 7. Control of Gluconeogenesis by Response Elements. Control of metabolism by response elements can be seen in the pathway of gluconeogenesis. What is gluconeogenesis? ○ Hepatic pathway whose major function is to maintain adequate glucose in the blood for tissues (e.g. nerves and RBC during fasting) ○ Provides glucose during periods of stress Glucagon and cortisol ○ Hormones that activate the pathway. ○ Stimulates PEPCK* gene. Gene coding for PEPCK enzyme What is PEPCK? MUTATION Enzyme that catalyzes a critical reaction in Module 1B #13, Gene Regulation and Mutation Video gluconeogenesis Lecture, 14:00-20:24 Under many conditions, it is the rate-limiting step Any permanent and heritable change in the DNA base of the pathway sequence of the organism is known as mutation. Effect of glucagon on PEPCK gene ○ Could be passed on to daughter cells if the mutated cell Induces expression of PEPCK gene divided Binds to a receptor in the cell membrane which increases Mutations can occur on a small scale, most often affecting concentration of cAMP one or two nucleotides of DNA, or they can involve large ○ Increase in concentration activates protein kinase A segments of a chromosome or an entire chromosome. which phosphorylates and activates cyclic adenosine The alteration in the DNA sequences can be reflected by the response element protein or CREB changes in the sequences of the bases in mRNA and (Prerequisite reading) sometimes by the changes in the amino acid sequence. 1. Glucagon binds to a receptor in the membrane. ○ Any mutation in a gene’s DNA can alter the function of the 2. cAMP has increased. protein encoded by the gene Regulation of Gene Torres 5 Expression and Mutation  Mutation can be changes in the enzyme activities, nutritional ○ Transversion requirements and therefore can sometimes cause genetic When a purine is replaced by a pyrimidine or vice diseases. versa (a pyrimidine is replaced by a purine) Cause antibiotic susceptibility, a change in morphology or a Single-base substitutions can also be classified into three change in many other properties of the cell. types (Types of Mutation-based Substitution): Mutation in germ cells are transmitted to the next progeny 1. Silent mutation and may give rise to inherited diseases Occurs if the new codon specifies the same amino Mutations in the somatic cells are not transmitted to the acid progeny but are important in the causation of cancer and NO detectable effect some congenital malfunctions. Ex: CGA to CGG = Arg to Arg (CGA and CGG specify Classification of Mutation: the same amino acid, Arginine) ○ Phenotypic Effects 2. Missense mutation ○ Type in Molecular Change Specifies a new amino acid Depending on the location of the missense amino acid I. CLASSIFICATION BASED ON PHENOTYPIC EFFECTS of the specific protein, the missense amino acid mutation may be acceptable, partially acceptable, LOSS-OF-FUNCTION MUTATION or unacceptable with respect to the function of the protein. Also known as null mutation or gene knockouts Ex: CGA to CCA = Arg to Pro Function of the gene product is eliminated. 3. Nonsense mutation Example: inability of the cell to make insulin. An amino acid codon becomes a STOP codon. Causes the premature termination of translation, GAIN-OF-FUNCTION MUTATION making the product usually nonfunctional. Give the gene product an increased activity or a new Ex: CGA to UGA = Arg to STOP function. Ex: Many human tumor carry missense mutation in the tp53 FRAMESHIFT MUTATION gene encoding for p53 ○ Here, aside from removing the tumor suppressor functions of the wild-type p53, the p53 mutations also give the mutant protein new activities that can contribute actively to various stages of tumor progression and increased resistance to anti-cancer treatment. The mutant p53 displays both loss-of-function and gain-of-function mutations. ○ WHY loss-of-function? Its activity that regulates the cell cycle and its function as tumor suppressor are abolished. II. CLASSIFICATION BASED ON TYPE OF MOLECULAR May be addition or deletion of 1 or more nucleotides that CHANGE generates altered mRNA ○ Results in an altered reading frame of the RNA POINT MUTATION Since there is no punctuation in the reading frame of the codon, the translation machinery does not recognize that a base was missing or added Result in the production of an entirely different protein after transcription and translation, sometimes, no protein at all Deletion : reading frame shifts to the left Addition : shifts to the right For more information on Mutations and examples of diseases caused by mutations, refer to the Module 1B #13 prerequisite readings uploaded on Moodle Figure X. Single-base substitutions (transitions and (pp.11-18). transversions) SUMMARY Involves: purines and pyrimidines Module 1B #13, Gene Regulation and Mutation Video ○ Purines are Adenine and Guanine (A and G) Lecture, 20:25-21:14 (end) ○ Pyrimidines are Thymine and Cytosine (T and C) Gene expression in eukaryotic organisms is controlled at A very common type of DNA-based alteration multiple levels and can occur in a variety of mechanisms. Includes: substitution, insertion, and deletion of a single Many of the important ones involve the regulation of base transcription. Only one base of the DNA is altered, which may be Mutation is a permanent and heritable change in the DNA transcribed into mRNA, and therefore, may result in the base sequence. This changed DNA sequence can be passed translation of a protein with abnormal amino acid sequences. on the mRNA and can also make a significant change in the Single-base substitutions may be transitions or polypeptide thus may cause genetic diseases. transversions. ○ Transition HORMONES when a purine is replaced by another purine (A to G or Online Video Lectures (Module 1B Regulation of Gene G to A), or a pyrimidine is replaced by another Expression) pyrimidine (T to C or C to T) Regulation of Gene Torres 6 Expression and Mutations  BIOCHEMISTRY REGULATION OF GENE EXPRESSION & MUTATIONS Sheila Marie P. Torres Trans Group: 1E, 2E Prerequisite readings and Handouts (Module 1B Regulation of Gene Expression) Regulation of Gene Torres 7 Expression and Mutation