AP Biology 2013

Chapter 18: Regulation of Gene Expression

Gene Regulation

✤ Prokaryotes and eukaryotes alter their gene expression in response to their changing environment

✤ In multicellular eukaryotes, gene expression regulates development and is responsible for differences in cell types

✤ RNA molecules play a role in regulating this

Bacteria

✤ Natural selection favors bacteria that produce only those products needed by the cell

✤ A cell can regulate the production of enzymes by feedback inhibition or by gene regulation

✤ In bacteria this is controlled by operons

Precursor Feedback inhibition

Enzyme 1

Enzyme 2

Enzyme 3

Tryptophan

(a) (b) Regulation of enzyme activity

Regulation of enzyme production

Regulation of gene expression

-

-

trpE gene

trpD gene

trpC gene

trpB gene

trpA gene

Fig. 18.2

1

2

3

Operons✤ Cluster of functionally related genes under coordinated control by a

single “on-off switch” that includes the operator, promoter, and genes they control

✤ Regulatory “switch” is a segment of DNA called an operator which is usually positioned within the promoter

✤ Operon can be switched off by a repressor protein (works by binding to the operator and blocking RNA polymerase)

✤ Repressor is the product of a separate regulatory gene

✤ Repressor can be in an active or inactive form depending on the presence of other molecules

✤ Corepressor is a molecule that cooperates with a repressor protein to shut off an operon

Ex. Trp Operon

✤ E. coli synthesizes the amino acid tryptophan

✤ Trp operon is on by default

✤ When tryptophan is present, it binds to the trp repressor protein which turns the operon off (acts as a corepressor)

Fig. 18.3

Promoter

DNA

Regulatory gene mRNA

trpR

5!

3!

Protein Inactive repressor

RNA polymerase

Promoter

trp operon

Genes of operon

Operator

mRNA 5!

Start codon Stop codon

trpE trpD trpC trpB trpA

E D C B A

Polypeptide subunits that make up enzymes for tryptophan synthesis

(a) Tryptophan absent, repressor inactive, operon on

(b) Tryptophan present, repressor active, operon off

DNA

mRNA

Protein

Tryptophan (corepressor)

Active repressor

No RNA made

Repressible and Inducible Operons

✤ Repressible operon is usually on (binding of a repressor shuts it off)

✤ Ex. trp operon

✤ Inducible operon is usually off (binding of an inducer inactivates the repressor and turns on transcription)

✤ Ex. lac operon - contains genes that code for enzymes used in hydrolysis and metabolism of lactose

(a) Lactose absent, repressor active, operon off

Regulatory gene

Promoter Operator

DNA lacZ lacI DNA

mRNA 5!

3!

No RNA made

RNA polymerase

Active repressor Protein

(b) Lactose present, repressor inactive, operon on

lacI

lac operon

lacZ lacY lacA DNA

mRNA 5!

3!

Protein

mRNA 5!

Inactive repressor

RNA polymerase

Allolactose (inducer)

β-Galactosidase Permease Transacetylase

Fig. 18.4

4

5

6

Positive Gene Regulation✤ Positive control caused by a stimulatory

protein called an activator of transcription (ex. catabolite activator protein - CAP)

✤ When glucose is scarce, CAP is activated by binding with cyclic AMP (cAMP)

✤ Activated CAP attaches to the promoter of the lac operon and increases the affinity of RNA polymerase (accelerating transcription)

✤ When glucose levels increase, CAP detaches from the lac operon and transcription returns to a normal rate

✤ CAP helps regulate other operons that encode enzymes used in catabolic pathways

Promoter

DNA

CAP-binding site

lacZ lacI

RNA polymerase binds and transcribes

Operator

cAMP Active CAP

Inactive CAP

Allolactose

Inactive lac repressor

(a) Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized

Promoter

DNA

CAP-binding site

lacZ lacI

Operator RNA polymerase less likely to bind

Inactive lac repressor

Inactive CAP

(b) Lactose present, glucose present (cAMP level low): little lac mRNA synthesized

Fig. 18.5

Eukaryote Gene Expression

✤ All organisms must regulate which genes are expressed at any given time

✤ In multicellular organisms, regulation of gene expression is essential for cell specialization

✤ Since almost all cells in an organism are genetically identical, differences between cell types result from differential gene expression (expression of different genes by cells with the same genome)

✤ Abnormalities in gene expression can lead to diseases including cancer

✤ Gene expression is regulated at many stages

Signal

NUCLEUS Chromatin

Chromatin modification: DNA unpacking involving histone acetylation and

DNA demethylation DNA

Gene

Gene available for transcription

RNA Exon Primary transcript

Transcription

Intron RNA processing

Cap Tail

mRNA in nucleus

Transport to cytoplasm

CYTOPLASM mRNA in cytoplasm

Translation Degradation of mRNA

Polypeptide Protein processing, such

as cleavage and chemical modification

Active protein Degradation

of protein Transport to cellular

destination

Cellular function (such as enzymatic activity, structural support)

Fig. 18.6

Regulation of Chromatin Structure

✤ Genes with highly packed heterochromatin are usually not expressed

✤ Chemical modifications to histones and DNA of chromatin influence both chromatin structure and gene expression

✤ Histone acetylation - acetyl groups are attached to positively charged lysines in histone tails which loosens chromatin structure (promoting transcription)

Amino acids available for chemical modification

Histone tails

DNA double helix

Nucleosome (end view)

(a) Histone tails protrude outward from a nucleosome

Unacetylated histones Acetylated histones (b) Acetylation of histone tails promotes loose chromatin

structure that permits transcription

Fig. 18.7

✤ Adding methyl groups (methylation) condenses chromatin

✤ If phosphate groups are added to the methylated amino acid, the chromatin is loosened.

7

8

9

DNA Methylation

✤ Adding methyl groups to certain bases in DNA is associated with reduced transcription in some species

✤ DNA methylation can cause long-term inactivation of genes in cellular differentiation

✤ In genomic imprinting, methylation regulates expression of either the maternal or paternal alleles of certain genes at the start of development

Epigenetic Inheritance

✤ Although chromatin modifications do not alter DNA sequences, they can be passed on to future generations of cells

✤ Inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence is called epigenetic inheritance

Organization of a Typical Eukaryotic Gene

✤ Most eukaryotic genes are associated with multiple control elements (segments of noncoding DNA that serve as binding sites for transcription factors that regulate transcription)

Enhancer (distal control

elements)

DNA

Upstream Promoter

Proximal control

elements Transcription

start site Exon Intron Exon Exon Intron

Poly-A signal

sequence Transcription termination

region

Downstream Poly-A signal

Exon Intron Exon Exon Intron

Transcription

Cleaved 3! end of primary transcript

5! Primary RNA transcript (pre-mRNA)

Intron RNA

RNA processing

mRNA

Coding segment

5! Cap 5! UTR Start

codon Stop

codon 3! UTR

3!

Poly-A tail

P P P G AAA ⋅⋅⋅ AAA

Fig. 18.8

10

11

12

Transcription Factors

✤ To initiate transcription, eukaryotic RNA polymerase requires the assistance of proteins called transcription factors (essential for transcription of all protein-coding genes)

✤ Proximal control elements are located close to the promoter. Distal control elements (called enhancers) may be far away from a gene or even located in an intron.

✤ An activator binds to an enhancer and stimulates transcription of a gene. (Activator has two domains - one binds to DNA and second activates transcription)

DNA

Activation domain

DNA-binding domain

Fig. 18.9

Transcription Factors

✤ Some act as repressors (inhibiting expression of a gene)

✤ Some activators and repressors act indirectly by influencing chromatin structure to promote or silence transcription

Activators DNA

Enhancer Distal control element

Promoter Gene

TATA box General transcription factors

DNA- bending protein

Group of mediator proteins

RNA polymerase II

RNA polymerase II

RNA synthesis Transcription initiation complex

Fig. 18.10

Post-Transcriptional Regulation✤ Transcription alone does not account

for gene expression

✤ Regulatory mechanisms can operate at various stages after transcription (allow cell to fine-tune gene expression rapidly in response to environmental changes)

✤ Alternative RNA Splicing - different mRNA molecules are produced from the same primary transcript (depending on what is treated as exons and introns)

✤ mRNA Degradation - life-span of mRNA molecule in cytoplasm (eukaryotic mRNA lasts longer than prokaryotic mRNA)

Exons

DNA

Troponin T gene

Primary RNA transcript

RNA splicing

or mRNA

1

1

1 1

2

2

2 2

3

3

3

4

4

4

5

5

5 5

Fig. 18.13

13

14

15

Post-Transcriptional Regulation

✤ Protein processing

✤ Proteasomes are complexes that bind protein molecules and degrade them

Protein to be degraded

Ubiquitin

Ubiquitinated protein

Proteasome

Protein entering a proteasome

Proteasome and ubiquitin to be recycled

Protein fragments (peptides)

Fig. 18.14

Noncoding RNA

✤ Small fraction of DNA codes for proteins and a very small fraction of non-protein-coding DNA consists of genes for rRNA or tRNA

✤ Significant amounts of the genome may be transcribed into noncoding RNAs (ncRNAs) which can regulate gene expression at mRNA translation and chromatin configuration

✤ MicroRNAs (miRNAs) are small single-stranded RNA that can bind to mRNA to degrade or block translation

(a) Primary miRNA transcript

Hairpin miRNA

miRNA

Hydrogen bond

Dicer

miRNA- protein complex

mRNA degraded Translation blocked (b) Generation and function of miRNAs

5! 3!

Fig. 18.15

Noncoding RNA

✤ Inhibition of gene expression by RNA molecules is called RNA interference (RNAi)

✤ RNAi is caused by small interfering RNAs (siRNAs) - siRNAs and miRNAs are similar but form from different RNA precursors

✤ An increase in the number of miRNAs in a species may have allowed morphological complexity to increase over evolutionary time

16

17

18

Differential Gene Expression

✤ During embryonic development, a fertilized egg gives rise to many different cell types which are organized into tissues, organs, organ systems, and the whole organism

✤ Transformation from a zygote to adult results from cell division, cell differentiation, and morphogenesis

✤ Cell differentiation - process by which cells become specialized in structure and function

✤ Morphogenesis - physical processes that give an organism its shape

✤ Cytoplasmic determinants - maternal substances in the egg that influence early development

(a) Cytoplasmic determinants in the egg (b) Induction by nearby cells

Unfertilized egg

Sperm

Fertilization

Zygote (fertilized egg)

Mitotic cell division

Two-celled embryo

Nucleus

Molecules of two different cytoplasmic determinants

Early embryo (32 cells)

NUCLEUS

Signal transduction pathway

Signal receptor

Signaling molecule (inducer)

(a) Fertilized eggs of a frog (b) Newly hatched tadpole

1 mm 2 mm

Figs. 18.16 & 18.17

Sequential Regulation

✤ Determination commits a cell to its final fate (happens before differentiation)

✤ Differentiation is marked by the production of tissue-specific proteins

✤ Ex. Myoblasts produce muscle-specific proteins and form skeletal muscle cells

✤ MyoD is one of several “master regulatory genes” the produce proteins that commit the cell to becoming skeletal muscle

✤ MyoD protein is a transcription factor that binds to enhancers of various target genes

Nucleus

Embryonic precursor cell

Myoblast (determined)

Part of a muscle fiber (fully differentiated cell)

DNA

Master regulatory gene myoD

OFF OFF

OFF mRNA

Other muscle-specific genes

MyoD protein (transcription factor)

mRNA mRNA mRNA mRNA

MyoD Another transcription factor

Myosin, other muscle proteins, and cell cycle– blocking proteins

Fig. 18.18

Setting Up the Body Plan

✤ Pattern formation - development of spatial organization of tissues and organs (in animals begins with establishment of major axes)

✤ Positional information - molecular cues that control pattern formation, tells a cell its location relative to the body axes and to neighboring cells

✤ Ex. Drosophila melanogaster (fruit fly) - cytoplasmic determinants in the egg determine the axes before fertilization

Head Thorax Abdomen

0.5 mm

BODY AXES

Anterior Left

Ventral

Dorsal Right

Posterior

(a) Adult

Egg developing within ovarian follicle

Follicle cell Nucleus

Nurse cell

Egg

Unfertilized egg Depleted nurse cells

Egg shell

Fertilization

Laying of egg Fertilized egg

Embryonic development

Segmented embryo

Body segments Hatching 0.1 mm

Larval stage

(b) Development from egg to larva

5

4

3

2

1

Fig. 18.19

19

20

21

Developmental Genes in Drosophila

✤ Homeotic genes - control pattern formation in late embryo, larva, and adult stages

✤ Embryonic lethals - mutations that cause death during embryogenesis

✤ Maternal effect genes - encode for cytoplasmic determinants that establish the axes (also called egg-polarity genes)

✤ Ex. Bicoid gene - if it is not functional, the fly will lack a front half and have duplicate posterior structures at both ends

Head Tail

Tail Tail

Wild-type larva

Mutant larva (bicoid)

250 µm

T1 T2 T3 A1 A2 A3 A4 A5 A6

A7 A8

A8 A7 A6 A7

A8

Fig. 18.21

Bicoid mRNA in mature unfertilized egg

Bicoid mRNA in mature unfertilized egg

Fertilization, translation of bicoid mRNA

Anterior end 100 µm

Bicoid protein in early embryo

Bicoid protein in early embryo

RESULTS

Fig. 18.22

Cancer✤ Gene regulation systems that go wrong in cancer are the same systems

involved in embryonic development

✤ Cancer is caused by mutations in genes that regulate cell growth and division

✤ Tumor viruses can also cause cancer in animals including humans

✤ Oncogenes - cancer-causing genes

✤ Proto-oncogenes - normal cellular genes responsible for normal cell growth and division

✤ Conversion of a proto-oncogene to an oncogene can lead to abnormal stimulation of the cell cycle

✤ Conversion can be caused by movement of DNA near a promoter, amplification of a proto-oncogene, point mutations in the proto-oncogene or its control elements

Conversion of a Proto-oncogene

Proto-oncogene

DNA

Translocation or transposition: gene moved to new locus, under new controls

Gene amplification: multiple copies of the gene

New promoter

Normal growth- stimulating protein in excess

Normal growth-stimulating protein in excess

Point mutation: within a control

element within

the gene

Oncogene Oncogene

Normal growth- stimulating protein in excess

Hyperactive or degradation- resistant protein

Fig. 18.23

22

23

24

Tumor Suppressor Genes

✤ Help prevent uncontrolled cell growth

✤ Mutations that decrease production of tumor suppressor genes contribute to cancer onset

✤ Tumor suppressor proteins repair damaged DNA, control cell adhesion, and inhibit the cell cycle

✤ Mutations in the ras proto-oncogene and p53 tumor suppressor gene are common in human cancers

✤ Mutations in the ras gene leads to hyperactive Ras protein and increased cell division

✤ p53 prevents a cell from passing on mutations due to DNA damage

Proto-oncogenes and Tumor Suppressor Genes

Growth factor

1

2

3

4

5

1

2

Receptor

G protein

Protein kinases (phosphorylation cascade) NUCLEUS

Transcription factor (activator)

DNA

Gene expression

Protein that stimulates the cell cycle

Hyperactive Ras protein (product of oncogene) issues signals on its own.

(a) Cell cycle–stimulating pathway

MUTATION

Ras

Ras

GTP

GTP

P P P P

P P

(b) Cell cycle–inhibiting pathway

Protein kinases

UV light

DNA damage in genome

Active form of p53

DNA

Protein that inhibits the cell cycle

Defective or missing transcription factor,

such as p53, cannot

activate transcription.

MUTATION

EFFECTS OF MUTATIONS

(c) Effects of mutations

Protein overexpressed

Cell cycle overstimulated

Increased cell division

Protein absent

Cell cycle not inhibited

3

Multistep Model of Cancer Development✤ Multiple mutations are generally needed for full-fledged cancer (this is why

incidence increases with age)

✤ Usually requires at least one active oncogene and mutations in several tumor-suppressor genes

✤ Individuals can inherit oncogenes or mutant alleles of tumor suppressor genesColon

Normal colon epithelial cells

Loss of tumor- suppressor gene APC (or other)

1

2

3

4

5 Colon wall

Small benign growth (polyp)

Activation of ras oncogene

Loss of tumor- suppressor gene DCC

Loss of tumor- suppressor gene p53

Additional mutations

Malignant tumor (carcinoma)

Larger benign growth (adenoma)

Fig. 18.25

25

26

27