Regulation of Gene ExpressionCh. 16.1-16.2;16.4-16.5

1 Embryo 200 Cell Types• From a single embryo, 200 types

of cells can be produced (differentiation)

• Diversity comes from genes being turned off

• Expression of the genes lead to specialization of the cell

• Transcriptional regulation controlling the expression of genes

1) post-transcriptional effect mRNA

2) Translational protein translation

3) Post-translational life span/activity of protein

Regulation in Prokaryotes• Adjust biochemistry quickly

as environment changes• Jacob and Monod

extensive studies into the effects of lactose on expression of lactase genes

• Operon regulatory sequence in DNA for a specific gene(s) + the genes

• Regulatory proteins bind to operons to promote or inhibit the transcription of transcription unit (single mRNA coded in the operon)

Regulation of an Operon• Operator section at the

start of the operon• Activator protein

attaches to operator to promote expression

• Repressor protein attaches to operator to inhibit expression

• Gene coding for regulatory proteins (activators/repressors) are called regulatory genes

• Non-regulating proteins come from structural genes

The lac Operon• 3 genes:1) lacZ codes for β-galactosidase;

breaks lactose into glucose + glactose

2) lacY codes for permease; actively transports lactose into the cell

3) lacA codes for transacetylase; We don’t know what it does

• Negatively regulated– Regulator gene lacI codes for Lac

repressor– Limits lac expression when lactose is

absent (normal)– When lactose is added, it is made into

allolactose (inducer for lac operon) – Inhibits lac repressor by binding to it

Lac Operon Part II; Positive Regulation• Lac operon is repressed in the

presence of lactose if glucose is also added. Why?– Glucose is a better source of energy– Converting lactose into usable

sugars (glucose) requires energy• CAP (catabolite activator protein)

activator synthesized in an inactive form; activated by cAMP (produced when glucose is absent)– Active form binds to CAP site at the

lac operon promoter allowing RNA Poly to attach

• If we add glucose, cAMP levels drop so CAP is deactivated and RNA Poly can bind to the DNA

trp Operon and Protein Synthesis• Some proteins, like

tryptophan, must be synthesized when not present to be absorbed

• trp Operon codes enzymes needed to make tryptophan; regulated by trpR (repressor) that is normally inactive; trp operon used to make tryptophan

• When tryptophan levels are high, the repressor is active and trp operon is blocked (repressible operon)

• Tryptophan is a corepressor; activates repressor

Regulation in Eukaryotes• Eukaryotes do not have

operons; regulatory gene are spread across the genome (side effect of variation)

• Eukaryotes use all forms of gene regulation:

1) Transcriptional Regulation

2) Post-transcriptional regulation

3) Translational regulation4) Post-translational

regulation

Transcriptional Regulation• Promoter region of DNA

upstream (~25bp) from the transcription unit– TATA Box 7-bp sequence

5’-TATAAAA-3’• TFs (transcription factors)

recognize TATA and bind to it; then RNA Poly II can bind

• Further upstream are the regulator sequences (promoter proximal elements) in the promoter proximal region

• Regulatory proteins bind here to enhance or repress transcription

Activators and Transcription• RNA Poly II + TFs transcription

initiation complex; not that efficient

• Activators proteins that help the complex attach and start translation

• Activators can be specific (one cell type for one gene) or general (multiple genes in all cell types) which are also called Housekeeping genes

• Enhancer regions on the DNA can increase transcription rate by interacting with activators (act as coactivators) by bending DNA into a loop

Motifs in DNA Binding Proteins• Domains structures in a protein made

from the combination of secondary folding options (helix, sheet, coil)– Ex. Helix-helix-coil-helix

• Motif specialized domains conserved in different types of proteins

• DNA interacting Motifs:1) Helix-turn-helix DNA binding region

of protein2) Zinc Finger finger shape with zinc

ion; bind to DNA grooves3) Leucine zipper dimers held together

by hydrophobic regions; bind to major groove of DNA

Combinational Gene Regulation• Regulation of most genes in more

complex than just activation or repression

• Genes can have multiple activators and repressors

• These regulation points between different genes overlap and follow the stronger influence

• Gene A is regulated by enhancer regions 1, 2 and 3; Gene B is regulated by enhancer 2, 3, and 4– Activators on 2 and 3 will produce A

and B proteins – Repressors on 3, and 4 will limit B

protein a great deal and A proteins a little bit

Coordinated Regulation• Proteins can be

regulated in complex organisms across many types of tissues through chemical signals (hormones)

• Steroid Hormone Response Element region in gene that hormone-receptor complex binds to– Allows regulation in

several cell types very quickly

Methylation of DNA• DNA methylation adding

methyl (-CH3) to cytosine bases– Turn off gene (silencing) by

blocking access to promoter region• Epigenetics change in gene

expression but no change in the DNA itself

• Hemoglobin turned off in all other cell types this way

• Genomic Imprinting silencing of one of two alleles during development– Methylated allele is not expressed

Chromatin Structure• Histones can block access

to DNA and thus regulate it

• Chromatin remodeling changing its structure– Nucleosome remodeling

complex moves histones along DNA or reshapes them to open a region

• Adding Acetyl Groups (CH3CO-) weakens the interactions between the histones and DNA

• Methylation of Histones marks histones wrapped with deactivated DNA

Gene Regulation in Development• Gene regulation is most

important during early development; determine the cell-types and physiology of the organism

• Regulation sensitive to both time (must all happen in the right order and within a certain window) and place (location in embryo determines location in body)

• Understanding comes from our model organisms:– Fruit fly, nematode worm,

zebrafish, and house mouse

From Zygote to Fetus• After fertilization, a zygote

develops into a fetus through several mechanisms

1) Mitosis need lots of cells2) Movement of cells cells need to

form the right shape3) Induction cell of a certain type

needs neighboring cells to respond to get a result

4) Determination totipotent cells becomes specific cell types

5) Differentiation cell types become finalized so tissue and systems can be made

Hold Up Mr. Nucleus…Cytoplasm has something to say…

• Not all regulation of a zygote comes from the nucleus

• Zygote’s cytoplasm is from the egg used at fertilization

• Cytoplasmic determinants– mRNA strands and proteins in

cytoplasm of egg also regulate the zygote

– Not reproduced during cell divisions; First divisions of zygote separate determinants asymmetrically so each daughter as an uncontrolled amount

– Only really take effect during the first few divisions but can last till tissues form

– Inherited only on the maternal side

Induction• Major step in the process of

determination• Signal molecules from very

specific cells (inducers) sent to receptor cells

• Two methods:1) Signal released and travels

short distances to receptors

2) Cell-to-Cell contact between proteins in the membranes of inducers and receptors

Differentiation• Determination narrows the

type of cells possible and differentiation limits to one cell type

• Genes required for cell type are left on while other genes are “turned off”

• Master regulatory genes promote the transcription of proteins needed to specialize the cell– myoD master gene

regulates MyoD transcription factors which promotes skeletal muscle proteins

Physical Position and Regulation• Pattern formation

arrangement of organs in the body– Discovered studying the

effects of mutations on the embryogenesis of fruit flies

– Particular genes control the body plan for all complex organism

• Steps required:1) Determine front, back,

head, and tail (ventral, dorsal, anterior, and posterior) of embryo

2) Divided zygote into segments

3) Use segments to map out body plan

Maternal-Effect Genes• Expressed when egg is

produced by the mother; mRNAs made from the bicoid gene

• Control the anterior-to-posterior polarity of the egg (front to back)

• Bicoid protein is produced and the highest conc. marks the anterior (head) and drops as move along to the posterior (butt) which has the lowest conc.

Segmentation Genes• 24 genes divide embryo

into regions• 3 Types:1) Gap Genes form

segments along A-P axis; broad regions

2) Pair-rule Genes divide broad regions with units of two segments each

3) Segment polarity Genes sets the boundaries for each segment; each segments needs an A-P axis

Homeotic Genes• Genes specify which

segment becomes what; where are the legs, eyes, wings, etc…– Hox genes– 8 Hox genes in fruit flies– Actually occur in order on

chromosome (AP)– Found in all animals and is

highly conserved• Homeo-Box region in all

homeotic genes that codes for its specific homeodomain (TF for its protein)

Genes and Cancer• 2 types of Cancer1) Familial Cancer inherited; common

with breast, colon, and testicular cancers

2) Sporadic Cancer occur randomly; more common form; can happen from viruses altering DNA

• All cancer is a multi-step process; need several key mutations

• 3 Classes of Genes effect cancer frequency:

1) Proto-oncogens2) Tumor suppressor genes3) microRNA genes

– (not covering this)

Proto-Oncogenes• Genes that stimulate cell

division in regular healthy cells• Code for growth factors, signal

receptors, transduction components, and TFs

• When mutated, they can become overactive oncogens

• Only one allele needs mutated to take effect– Mutation in the promoter – Mutation in the transcription unit– Translocation moves gene to a

more active promoter or enhancer

– Virus adds genes that activate or enhance a gene

Tumor Suppressor Genes• Code for proteins that inhibit

cell division• Keep Proto-oncogenes

repressed• TP53 codes for p53 that

inhibits CDKs used to pass the G1/S checkpoint

• If mutated, p53 can’t inhibit division

• p53 mutations are in 50% of all cancers

• Both alleles must be inactive for a tumor suppressor gene to lose function

Homework• Suggested Homework:– Test Your Knowledge Ch.

16• Actual Homework:– Discuss the Concepts #1– Interpret the Data Ch.

16– Design the Experiment

Ch. 16

Assignments for Next Week• PPT Presentations on Ch. 18:

– Groups of 3; 12-15 mins long– Topics:

• DNA Cloning and Building DNA Libraries• Gel Electrophoresis, Southern Blot, Northern Blot, and Western Blot• DNA Cloning and Bacteria Transformation for Protein Synthesis• BLAST Program and How it is Used

• Papers on Ch. 19:– 3 page paper discussing the following:

• Darwin’s Journey• Data and Experiments by Darwin• World Reaction to Darwin’s Theories• Basic Principles of Evolution

– DO NOT answer these section by section. These are the BIG IDEAS you paper must discuss. It should be a summary of Darwin’s life and impact on Biology