Regulation of Gene Expression Chapter 18

Gene expression Flow of genetic information Genotype to phenotype Genes to proteins Proteins not made at random Specific purposes Appropriate times

Control of gene expression Selective expression of genes All genes are not expressed at the same time Expressed at different times

Prokaryote regulation

Control of gene expression Regulate at transcription Gene expression responds to Environmental conditions Type of nutrients Amounts of nutrients Rapid turn over of proteins

(a) Regulation of enzyme activity (b) Regulation of enzyme production Fig. 18-2 Precursor Feedback inhibition trpE gene Enzyme 1 trpD gene Regulation of gene expression Enzyme 2 trpC gene trpB gene Enzyme 3 trpA gene Tryptophan (a) Regulation of enzyme activity (b) Regulation of enzyme production

Prokaryote Anabolism: Building up of a substance Catabolism: Breaking apart a substance

Prokaryote Operon Section of DNA Enzyme-coding genes Promoter Operator Sequence of nucleotides Overlaps promoter site Controls RNA polymerase access to the promoter

Prokaryote Multiple genes are expressed in a single gene expression trp operon Trytophan Synthesis Lac operon Lactose Degradation

Prokaryote trp Operon: Control system to make tryptophan Several genes that make tryptophan Regulatory region

Polypeptide subunits that make up enzymes for tryptophan synthesis Fig. 18-3a trp operon Promoter Genes of operon trpE trpD trpC trpB trpA Operator Start codon Stop codon mRNA 5 mRNA RNA polymerase 5 E D C B A Polypeptide subunits that make up enzymes for tryptophan synthesis

Prokaryote ⇧tryptophan present Bacteria will not make tryptophan Genes are not transcribed Enzymes will not be made Repression

Prokaryote Repressors Proteins Bind regulatory sites (operator) Prevent RNA polymerase attaching to promoter Prevent or decrease the initiation of transcription

Prokaryote Repressors Allosteric proteins Changes shape Active or inactive

Prokaryote ⇧tryptophan Tryptophan binds the trp repressor Repressor changes shape Active shape Repressor fits DNA better Stops transcription Tryptophan is a corepressor

(b) Tryptophan present, repressor active, operon off Fig. 18-3b-2 DNA No RNA made mRNA Protein Active repressor Tryptophan (corepressor) (b) Tryptophan present, repressor active, operon off

Prokaryote ⇩tryptophan Nothing binds the repressor Inactive shape RNA polymerase can transcribe

Polypeptide subunits that make up enzymes for tryptophan synthesis Fig. 18-3a trp operon Promoter Promoter Genes of operon DNA trpR trpE trpD trpC trpB trpA Regulatory gene Operator Start codon Stop codon 3 mRNA 5 mRNA RNA polymerase 5 E D C B A Protein Inactive repressor Polypeptide subunits that make up enzymes for tryptophan synthesis (a) Tryptophan absent, repressor inactive, operon on

Prokaryote Lactose Sugar used for energy Enzymes needed to break it down Lactose present Enzymes are synthesized Induced

Prokaryote lac Operon Promoter Operator Genes to code for enzymes Metabolize (break down) lactose

Prokaryote Lactose is present Repressor released Genes expressed Lactose absent Repressor binds DNA Stops transcription

Prokaryote Allolactose: Binds repressor Repressor releases from DNA Inducer Transcription begins Lactose levels fall Allolactose released from repressor Repressor binds DNA blocks transcription

(b) Lactose present, repressor inactive, operon on Fig. 18-4b lac operon DNA lacI lacZ lacY lacA RNA polymerase 3 mRNA mRNA 5 5 -Galactosidase Permease Transacetylase Protein Allolactose (inducer) Inactive repressor (b) Lactose present, repressor inactive, operon on

(a) Lactose absent, repressor active, operon off Fig. 18-4a Regulatory gene Promoter Operator DNA lacI lacZ No RNA made 3 mRNA RNA polymerase 5 Active repressor Protein (a) Lactose absent, repressor active, operon off

Prokaryote Lactose & tryptophan metabolism Adjustment by bacteria Regulates protein synthesis Response to environment Negative control of genes Operons turned off by active repressors Tryptophan repressible operon Lactose inducible operon

Prokaryote

Prokaryote Activators: Bind DNA Stimulate transcription Involved in glucose metabolism lac operon

Prokaryote Activator: Catabolite activator protein (CAP) Stimulates transcription of operons Code for enzymes to metabolize sugars cAMP helps CAP cAMP binds CAP to activate it CAP binds to DNA (lac Operon)

Prokaryote Glucose elevated cAMP low cAMP not available to bind CAP Does not stimulate transcription Bacteria use glucose Preferred sugar over others.

Prokaryote lac operon Regulated by positive & negative control Low lactose Repressor blocks transcription High lactose Allolactose binds repressor Transcription happens

Prokaryote lac operon Glucose also present CAP unable to bind Transcription will proceed slowly Glucose absent CAP binds promoter Transcription goes quickly

Eukaryote gene expression All cells in an organism have the same genes Some genes turned on Others remain off Leads to development of specialized cells Cellular differentiation

Eukaryote gene expression Gene expression assists in regulating development Homeostasis Changes in gene expression in one cell helps entire organism

Control of gene expression Chromosome structure Transcriptional control Posttranscriptional control

Fig. 18-6 Signal NUCLEUS Chromatin Chromatin modification DNA Gene available for transcription Gene Transcription RNA Exon Primary transcript Intron RNA processing Tail Cap mRNA in nucleus Transport to cytoplasm CYTOPLASM mRNA in cytoplasm Degradation of mRNA Translation Polypeptide Protein processing Active protein Degradation of protein Transport to cellular destination Cellular function

Eukaryotes 1. DNA is organized into chromatin 2. Transcription occurs in nucleus 3. Each gene has its own promoter

Chromatin structure DNA is tightly packaged Heterochromatin: Tightly packed Euchromatin: Less tightly packed Influences gene expression Promoter location Modification of histones

Chromatin structure Histone acetylation Acetyl groups (-COCH3) Attach to Lysines in histone tails Loosen packing Histone methylation Methyl groups (-CH3) Tightens packing

(a) Histone tails protrude outward from a nucleosome Fig. 18-7 Histone tails Amino acids available for chemical modification DNA double helix (a) Histone tails protrude outward from a nucleosome Unacetylated histones Acetylated histones (b) Acetylation of histone tails promotes loose chromatin structure that permits transcription

Chromatin structure Methylation of bases (cytosine) Represses transcription Embryo development

Eukaryotes Epigenetic change: Chromatin modifications Change in gene expression Passed on to the next generation Not a DNA sequence change

Transcription control RNA polymerase must bind DNA Proteins regulate by binding DNA RNA polymerase able to bind or not Stimulates transcription or blocks it

(distal control elements) Fig. 18-8-3 Poly-A signal sequence Enhancer (distal control elements) Proximal control elements Termination region Exon Intron Exon Intron Exon DNA Upstream Downstream Promoter Transcription Primary RNA transcript Exon Intron Exon Intron Exon Cleaved 3 end of primary transcript 5 RNA processing Intron RNA Poly-A signal Coding segment mRNA 3 Start codon Stop codon 5 Cap 5 UTR 3 UTR Poly-A tail

Eukaryotes Transcription RNA Polymerase Transcription factors (regulatory proteins) General transcription factors (initiation complex) Specific transcription factors

Eukaryotes Initiation of transcription Activator proteins Activator binds the enhancers Enhancers (DNA sequences) Interacts with the transcription factors Binds to the promoter RNA polymerase binds and transcription begins

Promoter Activators Gene DNA Enhancer Fig. 18-9-2 Promoter Activators Gene DNA Distal control element Enhancer TATA box General transcription factors DNA-bending protein Group of mediator proteins

Promoter Activators Gene DNA Enhancer Fig. 18-9-3 Promoter Activators Gene DNA Distal control element Enhancer TATA box General transcription factors DNA-bending protein Group of mediator proteins RNA polymerase II RNA polymerase II Transcription initiation complex RNA synthesis

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Enhancer Promoter Albumin gene Control elements Crystallin gene Fig. 18-10 Enhancer Promoter Albumin gene Control elements Crystallin gene LIVER CELL NUCLEUS LENS CELL NUCLEUS Available activators Available activators Albumin gene not expressed Figure 18.10 Cell type–specific transcription Albumin gene expressed Crystallin gene not expressed Crystallin gene expressed (a) Liver cell (b) Lens cell

Post transcriptional control RNA processing Primary transcript: Exact copy of the entire gene RNA splicing Introns removed from the mRNA snRNP’s (small nuclear ribonulceoproteins)

Post transcriptional control Splicing plays a role in gene expression Exons can be spliced together in different ways. Leads to different polypeptides Originated from same gene

Post transcriptional control Example in humans Calcitonin & CGRP Hormones released from different organs Derived from the same transcript

Exons DNA Troponin T gene Primary RNA transcript RNA splicing mRNA or Fig. 18-11 Exons DNA Troponin T gene Primary RNA transcript Figure 18.11 Alternative RNA splicing of the troponin T gene RNA splicing mRNA or

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Post transcriptional control Transport of transcript Passes through nuclear pores Active transport Cannot pass until all splicing is done

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Post transcriptional control Translation of RNA Translation factors are necessary Regulate translation Translation repressor proteins Stop translation Bind transcript Prevents it from binding to the ribosome

Post transcriptional control Ferritin (iron storage) Aconitase: Translation repressor protein Binds ferritin mRNA Iron will bind to aconitase Aconitase releases the mRNA Ferritin production increases

Post transcriptional control Protein modification Phosphorylation Other alterations can affect the activity of protein Insulin Starts out as a larger molecule Cut into more active sections

Post transcriptional control Protein modification Degradation Protein is marked by small protein Protein complex then breaks down proteins Proteasomes

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Protein processing and degradation Fig. 18-UN4 Chromatin modification Transcription • Genes in highly compacted chromatin are generally not transcribed. • Regulation of transcription initiation: DNA control elements bind specific transcription factors. • Histone acetylation seems to loosen chromatin structure, enhancing transcription. Bending of the DNA enables activators to contact proteins at the promoter, initiating transcription. • DNA methylation generally reduces transcription. • Coordinate regulation: Enhancer for liver-specific genes Enhancer for lens-specific genes Chromatin modification Transcription RNA processing • Alternative RNA splicing: RNA processing Primary RNA transcript mRNA degradation Translation mRNA or Protein processing and degradation Translation • Initiation of translation can be controlled via regulation of initiation factors. mRNA degradation • Each mRNA has a characteristic life span, determined in part by sequences in the 5 and 3 UTRs. Protein processing and degradation • Protein processing and degradation by proteasomes are subject to regulation.

Post transcriptional control Most gene regulation-transcription New discovery Small RNA’s 21-28 nucleotides long Play a role in gene expression New transcript before leaving the nucleus

Post transcriptional control RNA interference RNA forming double stranded loops from newly formed mRNA Loops are formed Halves have complementary sequences Loops inhibit expression of genes Where double RNA came from

Post transcriptional control Dicer: Cuts double stranded RNA into smaller RNA’s called microRNA (miRNA) Small interfering RNA (siRNA’s)

(b) Generation and function of miRNAs Fig. 18-13 Hairpin miRNA Hydrogen bond Dicer miRNA miRNA- protein complex 5 3 (a) Primary miRNA transcript Figure 18.13 Regulation of gene expression by miRNAs mRNA degraded Translation blocked (b) Generation and function of miRNAs

Post transcriptional control miRNA’s bind mRNA Prevents translation siRNA’s breaks apart mRNA before it’s translated

Post transcriptional control siRNAs play a role in heterochromatin formation Block large regions of the chromosome Small RNAs may also block transcription of specific genes

Chromatin modification Fig. 18-UN5 Chromatin modification • Small RNAs can promote the formation of heterochromatin in certain regions, blocking transcription. Chromatin modification Transcription Translation RNA processing • miRNA or siRNA can block the translation of specific mRNAs. mRNA degradation Translation Protein processing and degradation mRNA degradation • miRNA or siRNA can target specific mRNAs for destruction.

Embryonic development Zygote gives rise to many different cell types Cells →tissues → organs → organ systems Gene expression Orchestrates developmental programs of animals

(a) Fertilized eggs of a frog Fig. 18-14a Figure 18.14 From fertilized egg to animal: What a difference four days makes (a) Fertilized eggs of a frog

Embryonic development Zygote to adult results Cell division Cell differentiation: Cells become specialized in structure & function Morphogenesis: “creation of from” Body arrangement

(a) Fertilized egg (b) Four-cell stage (c) Early blastula Fig. 47-6 (a) Fertilized egg Figure 47.6 Cleavage in an echinoderm embryo (b) Four-cell stage (c) Early blastula (d) Later blastula

Fig. 47-1 Figure 47.1 How did this complex embryo form from a single cell? 1 mm

(a) 5 weeks (b) 14 weeks (c) 20 weeks Fig. 46-17 Figure 46.17 Human fetal development (a) 5 weeks (b) 14 weeks (c) 20 weeks

Embryonic development All cells same genome Differential gene expression Genes regulated differently in each cell type

Enhancer Promoter Albumin gene Control elements Crystallin gene Fig. 18-10 Enhancer Promoter Albumin gene Control elements Crystallin gene LIVER CELL NUCLEUS LENS CELL NUCLEUS Available activators Available activators Albumin gene not expressed Figure 18.10 Cell type–specific transcription Albumin gene expressed Crystallin gene not expressed Crystallin gene expressed (a) Liver cell (b) Lens cell

Embryonic development Specific activators Materials in egg cytoplasm Not homogeneous Set up gene regulation Carried out as cells divide

Embryonic development Cytoplasmic determinants Maternal substances in the egg Influence early development Zygote divides by mitosis Cells contain different cytoplasmic determinants Leads to different gene expression

(a) Cytoplasmic determinants in the egg Fig. 18-15a Unfertilized egg cell Sperm Nucleus Fertilization Two different cytoplasmic determinants Zygote Mitotic cell division Figure 18.15 Sources of developmental information for the early embryo Two-celled embryo (a) Cytoplasmic determinants in the egg

Embryonic development Environment around cell influences development Induction: Signals from nearby embryonic cells Cause transcriptional changes in target cells Interactions between cells induce differentiation of specialized cell types

(b) Induction by nearby cells Fig. 18-15b NUCLEUS Early embryo (32 cells) Signal transduction pathway Signal receptor Figure 18.15 Sources of developmental information for the early embryo Signal molecule (inducer) (b) Induction by nearby cells

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Embryonic development Determination: Observable differentiation of a cell Commits a cell to its final fate Cell differentiation is marked by the production of tissue-specific proteins Gives cell characteristic structure & function

Embryonic development Myoblasts: Produce muscle-specific proteins Form skeletal muscle cells MyoD One of several “master regulatory genes” Produces proteins Commit cells to becoming skeletal muscle

Embryonic development MyoD protein Transcription factor Binds to enhancers of various target genes Causes expression

Master regulatory gene myoD Other muscle-specific genes Fig. 18-16-1 Nucleus Master regulatory gene myoD Other muscle-specific genes DNA Embryonic precursor cell OFF OFF Figure 18.16 Determination and differentiation of muscle cells

MyoD protein (transcription Myoblast factor) (determined) Nucleus Fig. 18-16-2 Nucleus Master regulatory gene myoD Other muscle-specific genes DNA Embryonic precursor cell OFF OFF mRNA OFF MyoD protein (transcription factor) Myoblast (determined) Figure 18.16 Determination and differentiation of muscle cells

(fully differentiated cell) Fig. 18-16-3 Nucleus Master regulatory gene myoD Other muscle-specific genes DNA Embryonic precursor cell OFF OFF mRNA OFF MyoD protein (transcription factor) Myoblast (determined) Figure 18.16 Determination and differentiation of muscle cells mRNA mRNA mRNA mRNA Myosin, other muscle proteins, and cell cycle– blocking proteins MyoD Another transcription factor Part of a muscle fiber (fully differentiated cell)

Embryonic development Pattern formation: Development of spatial organization of tissues & organs Begins with establishment of the major axes Positional information: Molecular cues control pattern formation Tells a cell its location relative to the body axes & neighboring cells

Fruit fly Unfertilized egg contains cytoplasmic determinants Determines the axes before fertilization After fertilization, Embryo develops into a segmented larva with three larval stages

Head Thorax Abdomen 0.5 mm Dorsal Right BODY AXES Anterior Posterior Fig. 18-17a Head Thorax Abdomen 0.5 mm Dorsal Right BODY AXES Anterior Posterior Figure 18.17 Key developmental events in the life cycle of Drosophila Left Ventral (a) Adult

(b) Development from egg to larva Fig. 18-17b Follicle cell 1 Egg cell developing within ovarian follicle Nucleus Egg cell Nurse cell 2 Unfertilized egg Egg shell Depleted nurse cells Fertilization Laying of egg 3 Fertilized egg Embryonic development Figure 18.17 Key developmental events in the life cycle of Drosophila 4 Segmented embryo 0.1 mm Body segments Hatching 5 Larval stage (b) Development from egg to larva

Fruit fly Homeotic genes: Control pattern formation in late embryo,larva and adult

Eye Leg Antenna Wild type Mutant Fig. 18-18 Figure 18.18 Abnormal pattern formation in Drosophila Wild type Mutant

Fruit fly Maternal effect genes: Encode for cytoplasmic determinants Initially establish the axes of the body of Drosophila Egg-polarity genes: Maternal effect genes Control orientation of the egg Consequently the fly

Fruit Fly Bicoid gene Maternal effect gene Affects the front half of the body An embryo whose mother has a mutant bicoid gene Lacks the front half of its body Duplicate posterior structures at both ends

EXPERIMENT Tail Head Wild-type larva Tail Tail Mutant larva (bicoid) Fig. 18-19a EXPERIMENT Tail Head A8 T1 T2 T3 A7 A1 A6 A2 A3 A4 A5 Wild-type larva Tail Tail Figure 18.19 Is Bicoid a morphogen that determines the anterior end of a fruit fly? A8 A8 A7 A7 A6 Mutant larva (bicoid)