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Chapter 13 Regulation of Gene Activity • Humans and nemotodes have about the same number of genes roughly 20,500 • So how can a complex organism produce the proteins they require? • By regulation of pre-mRNA splicing to produce many proteins from a single gene • In 1961, Jocob and Monod showed that the bacteria Escherichia coli could regulate the expression of genes • They received the Nobel prize for the “operon model” to express gene regulation in prokaryotes • Operon includes: • Promoter which is a short sequence of DNA where RNA polymerase first attaches to begin transcription • Operator which is a short portion of DNA where an active repressor binds • When active repressor binds to operator, RNA polymerase cannot attach to promoter and no transcription • Structural genes are one to several genes coding for primary structure of enzymes in metabolic pathway transcribed as a unit • Regulator gene, usually located outside operon and controlled by own promoter, codes for a repressor that controls whether the operator is active or not -Some operons in E. coli are usually in “on” rather than “off” condition -Trp operon, the regulator codes a repressor that ordinarily is unable to attach to operator -RNA polymerase is able to bind to promoter and structural genes are expressed -Then five enzymes promote anabolic pathway for synthesis of amino acid tryptophan -If tryptophan is present, it binds to the repressor, changes its shape and binds to operon Structural genes are not expressed • Entire unit is called a repressor operon • Tryptophan is the corepressor • Repressible operons are usually involved in anobolic pathways Fig. 13.1 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. regulator gene promoter operator structural genes DNA RNA polymerase 5 3 mRNA inactive repressor enzymes a. Tryptophan absent. Enzymes needed to synthesize tryptophan are produced. RNA polymerase cannot bind to promoter. DNA active repressor tryptophan inactive repressor b. Tryptophan present. Presence of tryptophan prevents production of enzymes used to synthesize tryptophan. • Bacteria metabolism is efficient • If a protein or enzyme is not needed, genes to make them are inactive • If lactose is not present, enzymes for lactose catabolism are not active • If E coli are denied glucose and given lactose, it immediately makes the three enzymes needed to metabolize lactose • The three structural genes needed are adjacent to one another and under control of a single promoter and operon • Lac operator repression usually binds to operator and prevents transcription • Lactose binds to repressor, changes its shape that prevents its binding to promoter • RNA polymerase binds to promoter and carries out transcription of enzymes for lactose metabolism • Presence of lactose brings about expression of genes and is called inducer • Entire unit is called inducible operon • Inducible operons are usually necessary for catabolic pathways Fig. 13.2 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. RNA polymerase cannot bind to promoter. regulator gene promoter operator structural genes DNA active repressor active repressor a. Lactose absent. Enzymes needed to take up and use lactose are not produced. RNA polymerase can bind to promoter. DNA inactive repressor 5 mRNA active repressor lactose enzymes b. Lactose present. Enzymes needed to take up and use lactose are produced only when lactose is present. 3 • E coli prefers using glucose • A molecule called cyclic AMP (cAMP) accumulates when glucose is absent • cAMP binds to a molecule called catabolite activator protein (CAP) and the complex attaches to site next to lac promoter • This bends DNA exposing the promoter to RNA polymerase Page 236 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. adenine 5 P O CH2 3 OH cyclic AMP (cAMP) Fig. 13.3 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. CAP binding site promoter operator DNA RNA polymerase binds fully with promoter. cAMP active CAP inactive CAP a. Lactose present, glucose absent (cAMP level high) CAP binding site promoter operator DNA RNA polymerase does not bind fully with promoter. inactive CAP b. Lactose present, glucose present (cAMP level low) • Each cell in multicellular eukaryote, has a copy of all genes • Different genes are actively expressed in different cells • Types of control in eukaryotic cells: • 1) Chromatin structure- Chromatin packing is used to keep genes turned off by preventing access to RNA polymerase • In nucleus, loosely condensed chromatin is available for transcription • Part of epigenetic inheritance, the transcription of genetic information outside coding sequence of a gene • 2) Transcriptional control is the degree to which a gene is transcribed into mRNA determines amount of gene product • Transcription factors may promote or repress transcription • 3) Posttranscriptional control involves mRNA processing and how fast mRNA leaves the nucleus • Can determine type of protein product made and amount of gene product made in a given time • 4) Translational control occurs in cytoplasm and affects when translation begins and how long it continues • Any influence on the persistence of 5’ cap and 3’ poly-A tail affect length of translation • Excised introns are involved in regulatory system and affect life span of mRNA • 5) Posttranslational control occurs in cytoplasm after protein synthesis • Only functional protein is an active gene product Fig. 13.4 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. histones Chromatin structure Transcriptional control 3 premRNA 5 intron exon Posttranscriptional control mRNA 5 3 nuclear pore nuclear envelope Translational control polypeptide chain Posttranslational control plasma membrane functional protein • Highly condensed heterochromatin is inaccessible to RNA polymerase • It appears as darkly stained portions within nucleus in electron micrographs • Example of heterochromatin is the Barr body in mammalian females • It is an inactive X chromosome that does not produce gene products • In females one X chromosome transcribes genes and the other becomes a Barr body • Which X is inactive depends on which X chromosome that cell received • One X comes from father and the other from the mother • Conditions in human females include: ocular albinism, Duchanne muscular distrophy, X-linked hereditory absence of sweat glands Fig. 13.6 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Coats of tortoiseshell cats have patches of orange and black. active X chromosome allele for orange color inactive X Barr bodies cell division inactive X allele for black color active X chromosome Females have two X chromosomes. One X chromosome is inactivated in each cell. Which one is by chance. © Chanan Photo 2004 • Term euchromatin is used for the more loosely packed active chromatin • In herterochromatin, the histone tails tend to bear methyl groups (-CH3) • In euchromatins, the histone tails tend to be acetylated and have attached acetyl groups (-COCH3) • When euchromatin is transcribed, chromatin remodeling complex pushes aside the histone portion of nucleosome so access to DNA is not barred • Also affects gene expression by adding acetyl or methyl groups to histone tails • Epigenetic inheritance concerns the pattern of inheritance that does not depend on only the genes • If a histone is methylated, the DNA may also be methylated • Genomic imprinting occurs when either the mother’s or father’s allele is methylated during gamete formation • If inherited, the gene is not expressed • Transcriptional control is the most critical of all controls • No operons like those in prokaryotic cells have been found in eukaryotes • Every cell contains transcriptional factors, proteins that help regulate transcription • In eukaryotes, transcription activators are DNA binding proteins that speed transcription • They bind to a region of DNA called enhancer that can be far away from promoter • A hairpin loop in the DNA brings the transcription activators attached to enhancers into contact with transcriptional factor complex • Transcription factors, activators,and repressors are always present in nucleus, but have to be activated before they bind to DNA Fig. 13.7 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. DNA promoter enhancer gene transcription activator mediator proteins transcription factor complex RNA polymerase mRNA transcription • During pre-mRNA splicing, introns (noncoding regions) are excised, and exons (expressed regions) are joined together to form mRNA • Sometimes an exon is skipped or an intron is included • Results in mature mRNA that has an altered sequence, and protein encoded differs Fig. 13.8 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. intron 5 cap A exon B C D pre-mRNA RNA intron E 3 5 poly-A tail cap B C D pre-mRNA splicing RNA intron mRNA mRNA protein product 1 protein product 2 3 poly-A tail intron A B D E b. E splicing C A B CDE a. A exon • Translation control begins when processed mRNA reaches cytoplasm and before there is a protein product • Includes presence or absence of 5’ cap and length of poly-A tail at 3’ end • Micro RNAs (miRNAs) can regulate translation by causing the destruction of mRNAs before they can be translated • Much like a dimmer switch on a light, miRNAs can fine-tune the expression of genes Fig. 13.9 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. pre-mRNA 5 3 microRNA (miRNA) MicroRNA is cut from a pre-mRNA and binds with proteins to form RISC. 3 5 RISC (RNA-induced silencing complex) proteins Complementary base pairing between RNAs allows RISC to bind to mRNA. mRNA 5 3 RISC Translation is inhibited. or The mRNA is degraded. • A gene mutation is a permanent change in the sequence of bases in DNA • Can range from no effect to complete inactivation • Germ-line mutations occur in sex cells and can be passed to subsequent generations • Somatic mutations occur in body cells and affect only a small number of cells in a tissue • Somatic mutations are not passed on to future generations, but can lead to cancer • Spontaneous mutations are associated with any number of normal processes • The movement of transposons from one chromosomal • location to another can disrupt a gene and lead to an abnormal product • A base in DNA may undergo a chemical change that leads to a miss pairing during replication • These mutations are rare because DNA polymerase proofreads the new strand against the old strand, detects most mismatched nucleotides, and usually replaces them with correct nucleotides • Induced mutations are caused by mutagens, environmental factors that can alter base composition of DNA • Includes radiation and organic chemicals • Many mutations are also carcinogens (cancer-causing) • Chemical mutagens are present in some food we eat and many industrial chemicals • Tobacco smoke contains a number of carcinogenic organic chemicals • One-third of all cancer deaths can be attributed to smoking • Lung cancer is most frequent lethal cancer in the United States • Ames test is used for mutagenicity of a chemical to be carcinogenic • A histidine-requiring strain of bacteria is exposed to • the chemical • If the chemical is mutagenic, bacteria can grow without histidine Fig. 13.10 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Suspected chemical mutagen Control bacterial strain (requires histidine) Plate onto petri plates that lack histidine. bacterial growth bacterial strain (requires histidine) Incubate overnight Mutation occurred Mutation did not occur • Point mutations involve a change in a single DNA nucleotide with a possible change in a specific amino acid • Frameshift mutations occur most often because one or more nucleotides are either inserted or deleted from DNA • May form a completely new sequence of codons and nonfunctioning protein • A single nonfunctioning protein can have a dramatic effect on the phenotype, because enzymes are often part of metabolic pathways Page 244 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. A (phenylalanine) EA B (tyrosine) EB C (melanin) Fig. 13.12 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5 3 No mutation C A C G T GG AGT G AG G T C T C C T C Val His His Leu Thr Pro Glu Glu His (normal protein) C A C G T A G AG T G A G G T C T C C T C Val His Leu Thr Pro Glu Glu b. Normal red blood cell Glu Val (abnormal protein) C A C G T GG AGT G AG G T C A C C T C Val Glu Stop (incomplete protein) a. His Leu Thr Pro Val Glu C A C G T GG AGT G AG G T A T C C T C Val His Leu Thr Pro Stop b, c: © Stan Flegler/Visuals Unlimited. c. Sickled red blood cell • The development of cancer involves a series of accumulating mutations that can be different for each type of cancer • The cell cycle occurs inappropriately when protooncogenes become oncogenes and tumor suppressor genes are no longer effective