Biology Sylvia S. Mader Michael Windelspecht Chapter 13 Regulation of Gene Expression Lecture Outline See separate FlexArt PowerPoint slides for all figures and tables pre-inserted into PowerPoint without notes. 1 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 1

Outline 13.1 Prokaryotic Regulation 13.2 Eukaryotic Regulation 13.3 Genetic Mutations

13.1 Prokaryotic Regulation Bacteria don’t require same enzymes all the time Enzymes produced as needed Operon model: (François Jacob & Jacques Monod 1961) explains prokaryotic regulation of gene expression Operon: group of structural & regulatory genes functioning as 1 unit

Prokaryotic Regulation An operon consists of three components Promoter Short DNA sequence where RNA polymerase first attaches Operon start, transcription begins Operator Short DNA sequence where active repressor binds RNA polymerase can’t bind, no transcription Structural Genes >1 genes coding for enzymes/proteins of metabolic pathway Transcribed as a block Long segment of DNA Regulatory gene- codes for repressor protein located outside the operon repressor protein controls whether operon is active or not

Prokaryotic Regulation The trp Operon Regulator genes code for inactive repressor If tryptophan is absent: Repressor unable to attach to operator (expression is “on”) RNA polymerase binds to promoter Enzymes for tryptophan synthesis are produced If tryptophan is present: Combine w/repressor protein as co-repressor Repressor binds operator, RNA polymerase can’t bind to promoter Repressor blocks synthesis of structural genes for tryptophan synthesis

transcription is prevented. The trp Operon Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. promoter operator regulator gene structural genes When the repressor binds to the operator, transcription is prevented. active repressor

The trp Operon regulator gene promoter operator structural genes DNA Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. regulator gene promoter operator structural genes DNA RNA polymerase 5′ 3′ mRNA X mRNA inactive repressor enzymes a. Tryptophan absent. Enzymes needed to synthesize tryptophan are produced. RNA polymerase cannot bind to promoter. DNA active repressor mRNA tryptophan inactive repressor b. Tryptophan present. Presence of tryptophan prevents production of enzymes used to synthesize tryptophan.

Prokaryotic Regulation The lac Operon Regulator genes code for active repressor If lactose is absent: Repressor attaches to operator RNA polymerase can’t bind to promoter Expression is normally “off” If lactose is present: combines w/ repressor; can’t bind to operator RNA polymerase binds to promoter 3 enzymes necessary for lactose catabolism are produced

The lac Operon a. b. regulatory gene promoter operator Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. regulatory gene promoter operator lactose metabolizing genes DNA mRNA RNA polymerase active repressor a. RNA polymerase bound to promoter. DNA x mRNA inactive repressor lactose active repressor enzymes mRNA b.

Prokaryotic Regulation Further control of the lac operon E. coli preferentially breaks down glucose lac operon is maximally activated only in the absence of glucose When glucose is absent Cyclic AMP (cAMP) accumulates cAMP binds to catabolite activator protein (CAP) CAP + cAMP, binds to site near lac promoter  RNA polymerase better able to bind to promoter structural genes of the lac operon expressed more efficiently 10

Prokaryotic Regulation Further control of the lac operon When glucose is present little cAMP in cell CAP is inactive lac operon is not expressed maximally 11

Action of CAP a. Lactose present, glucose absent (cAMP level high) Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. CAP binding site promoter operator DNA inactive CAP 12 a. Lactose present, glucose absent (cAMP level high)

Action of CAP a. Lactose present, glucose absent (cAMP level high) Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. CAP binding site promoter operator DNA cAMP inactive CAP 13 a. Lactose present, glucose absent (cAMP level high)

Action of CAP a. Lactose present, glucose absent (cAMP level high) Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. CAP binding site promoter operator DNA cAMP inactive CAP 14 a. Lactose present, glucose absent (cAMP level high)

Action of CAP a. Lactose present, glucose absent (cAMP level high) Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. CAP binding site promoter operator DNA cAMP active CAP inactive CAP 15 a. Lactose present, glucose absent (cAMP level high)

Action of CAP a. Lactose present, glucose absent (cAMP level high) Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. CAP binding site promoter operator DNA cAMP active CAP inactive CAP 16 a. Lactose present, glucose absent (cAMP level high)

Action of CAP a. Lactose present, glucose absent (cAMP level high) 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 17 a. Lactose present, glucose absent (cAMP level high)

Action of CAP 18 CAP binding site promoter operator DNA 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 18 b. Lactose present, glucose present (cAMP level low)

13.2 Eukaryotic Regulation A variety of mechanisms 5 primary levels of control: Nuclear levels Chromatin Structure Transcriptional Control Posttranscriptional Control Cytoplasmic levels 4. Translational Control 5. Posttranslational Control

Control of Gene Expression in Eukaryotic Cells Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. histones Chromatin structure Transcriptional control 3 pre- mRNA 5 intron exon Posttranscriptional control mRNA 5 3 nuclear pore nuclear envelope Translational control polypeptide chain Posttranslational control plasma membrane functional protein

Eukaryotic Regulation 1. Chromatin Structure Chromatin= Eukaryotic DNA + histone proteins Nucleosomes DNA wound around 8 molecules of histone proteins Looks like beads on a string levels of chromatin packing determined by degree of nucleosome coiling

Chromatin Structure Regulates Gene Expression Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. nucleolus heterochromatin euchromatin nucleosome inaccessible promoter 1 µm a. Darkly stained heterochromatin and lightly stained euchromatin chromatin remodeling complex H2B histone protein H2A H4 histone tail H3 accessible promoter DNA H1 DNA to be transcribed b. A nucleosome c. DNA unpacking (a): © Dennis Kunkel Microscopy,Inc./Visuals Unlimited

Eukaryotic Regulation 1. Chromatin Structure cont. Euchromatin Loosely coiled DNA Transcriptionally active Heterochromatin Tightly packed DNA Transcriptionally inactive Barr Body Females have 2 X chromosomes, only one is active other X chromosome is tightly packed & is inactive inactive X chromosome is a Barr body

X-Inactivation in Mammalian Females 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

Eukaryotic Regulation 2. Transcriptional Control- most critical level! Transcription controlled by transcription factors (proteins) Transcription activator bind to enhancer DNA Regions of DNA where factors that regulate transcription can also bind Transcription factors are always present in the cell, but most likely have to be activated before they will bind to DNA

Eukaryotic Transcription Factors Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. DNA promoter gene enhancer transcription activator transcription factor complex mediator proteins RNA polymerase mRNA transcription

Eukaryotic Regulation 3. Posttranscriptional control operates on primary mRNA transcript Alter pre-mRNA transcript: Excision of introns can vary Splicing of exons can vary Determines type of mature transcript that leaves nucleus Control speed of mRNA transport from nucleus Affect # of transcripts arriving at rough ER & amount of gene produced

Alternative Processing of pre-mRNA Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. intron exon intron exon 5 A B C D E 3 5 A B C D E 3 cap pre-mRNA poly-A tail cap pre-mRNA poly-A tail RNA splicing RNA splicing intron intron C A B C D E A B D E mRNA mRNA protein product 1 protein product 2 a. b.

Eukaryotic Regulation 4. Translational Control – Determines degree of mRNA translated into protein Features of mRNA affect whether translation occurs and how long mRNA is active Presence of 5′ cap Length of poly-A tail on 3′ end MicroRNAs (miRNAs) regulate translation by causing destruction of mRNAs before they are translated

Eukaryotic Regulation 5. Posttranslational Control – Affects activity of protein product accomplished by regulating: Activation Degradation rate 30

13.3 Gene Mutations gene mutation- permanent change in DNA sequence gene mutation effects No effect  Complete protein inactivation Germ-line mutations occur in sex cells Somatic mutations occur in body cells

Gene Mutation Causes 1. Spontaneous mutations Chemical changes in DNA leads to mispairing during replication Movement of transposons from one location to another Replication Errors- rare! DNA polymerase Proofreads new strands Generally corrects errors Overall mutation rate= 1 in 1,000,000,000 nucleotide pairs replicated

Gene Mutation Causes 2. Induced mutations Caused by mutagens Many mutagens are carcinogens (cancer-causing) Chemical Mutagens- food additives! Ultraviolet Radiation- the sun, x-rays, gamma rays, can repair damage (DNA repair enzymes) Organic Chemicals- tobacco smoke  cancers!!

The Ames Test For Mutagenicity Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Suspected chemical mutagen Control bacterial strain (requires histidine) bacterial strain (requires histidine) Plate onto petri plates that lack histidine. bacterial growth Incubate overnight Mutation occurred Mutation did not occur

Gene Mutations Point Mutations Frameshift Mutations Involve change in 1 DNA nucleotide Change 1 codon to different codon Effects on protein vary: Nonfunctional Reduced functionality Unaffected Frameshift Mutations 1-2 nucleotides inserted or deleted from DNA Protein always rendered nonfunctional Normal : THE CAT ATE THE RAT After deletion: THE ATA TET HER AT After insertion: THE CCA TAT ETH ERA T

Point Mutations in Hemoglobin Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 3 5 No mutation A C A C G T G G G T G A G G T C T C C T C Val His Leu Thr Pro Glu Glu b. Normal red blood cell c. Sickled red blood cell b, c: © Stan Flegler/Visuals Unlimited.

Point Mutations in Hemoglobin Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 3 5 No mutation C A C G T G G A G T G A G G T C T C C T C Val His Leu Thr Pro Glu Glu His His (normal protein) C A C G T A G A G T G A G G T C T C C T C Val His Leu Thr Pro Glu Glu b. Normal red blood cell c. Sickled red blood cell b, c: © Stan Flegler/Visuals Unlimited. 37

Point Mutations in Hemoglobin Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 3 5 No mutation C A C G T G G A G T G A G G T C T C C T C Val His Leu Thr Pro Glu Glu His His (normal protein) C A C G T A G A G T G A G G T C T C C T C Val His Leu Thr Pro Glu Glu b. Normal red blood cell c. Sickled red blood cell Glu Val (abnormal protein) C A C G T G G A G T G A G G T C A C C T C Val His Leu Thr Pro Val Glu b, c: © Stan Flegler/Visuals Unlimited. 38

Point Mutations in Hemoglobin Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 3 5 No mutation C A C G T G G A G T G A G G T C T C C T C Val His Leu Thr Pro Glu Glu His His (normal protein) C A C G T A G A G T G A G G T C T C C T C Val His Leu Thr Pro Glu Glu b. Normal red blood cell c. Sickled red blood cell Glu Val (abnormal protein) C A C G T G G A G T G A G G T C A C C T C Val His Leu Thr Pro Val Glu Glu Stop (incomplete protein) C A C G T G G A G T G A G G T A T C C T C Val His Leu Thr Pro Stop a. b, c: © Stan Flegler/Visuals Unlimited. 39

Gene Mutations Cancer development involves series of accumulating mutations Proto-oncogenes – Stimulate cell division (gas pedal) Mutated proto-oncogenes  oncogenes that’re always active Tumor suppressor genes – inhibit cell division (brakes) Mutations in oncogene & tumor suppressor genes: Stimulate cell cycle uncontrollably Lead to tumor formation

Cell Signaling Pathway That Stimulates a Mutated Tumor Suppressor Gene Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. receptor plasma membrane nucleus mutated tumor suppressor gene

Cell Signaling Pathway That Stimulates a Mutated Tumor Suppressor Gene Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. inhibiting growth factor receptor plasma membrane nucleus mutated tumor suppressor gene 42

Cell Signaling Pathway That Stimulates a Mutated Tumor Suppressor Gene Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. inhibiting growth factor receptor plasma membrane signal transducers nucleus mutated tumor suppressor gene 43

Cell Signaling Pathway That Stimulates a Mutated Tumor Suppressor Gene Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. inhibiting growth factor receptor plasma membrane signal transducers transcription factor nucleus mutated tumor suppressor gene 44

Cell Signaling Pathway That Stimulates a Mutated Tumor Suppressor Gene Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. inhibiting growth factor receptor plasma membrane signal transducers transcription factor protein that is unable to inhibit the cell cycle or promote apoptosis nucleus mutated tumor suppressor gene 45

Cell Signaling Pathway That Stimulates an Oncogene Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. receptor plasma membrane cytoplasm nucleus oncogene

Cell Signaling Pathway That Stimulates an Oncogene Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. stimulating growth factor receptor plasma membrane cytoplasm nucleus oncogene 47

Cell Signaling Pathway That Stimulates an Oncogene Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. stimulating growth factor receptor plasma membrane signal transducers cytoplasm nucleus oncogene 48

Cell Signaling Pathway That Stimulates an Oncogene Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. stimulating growth factor receptor plasma membrane signal transducers cytoplasm transcription factor nucleus oncogene 49

Cell Signaling Pathway That Stimulates an Oncogene Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. stimulating growth factor receptor plasma membrane signal transducers cytoplasm transcription factor protein that overstimulates the cell cycle nucleus oncogene 50