Chapter 18 Regulation of Gene Expression. Genetic Diversity in Asexual Bacteria? I. From Within A....

Chapter 18

Regulation of Gene

Expression

Genetic Diversity in Asexual Bacteria?

I. From Within

A. High mutation rate

B. Transposons

II. Recombination

A. Transformation

B. Transduction

C. Conjugation

I. Bacterial Regulation

A. Advantage to expressing only the genes necessary?

B. Metabolic control

1. Adjust enzyme activity

2. Adjust enzyme production

Gene expression control


C. The Operon Model

1. Discovery – Jacob and Monod, 1961

2. The Basic Concept

Operon – A cluster of genes related to the same pathway

See E.coli – Tryptophan example (352)

I. Bacterial Regulation2. The Basic Concept

Operon

a. Components;

A Single promoter sequence

A Single operator sequence

Multiple genes

(One for each protein in the pathway)

I. Bacterial Regulation2. The Basic ConceptOperon

b. Functions as a single transcription unitWith a single “on-off” switchAllows coordination and control


2. The Basic ConceptOperon

c. “On-off” switch = OperatorSegment of DNA

Upstream of the genesWithin the promotorContols access of RNA

Polymerase to the genes


2. The Basic Concept

Operon

d. Mechanism? (Example – Trp operon)

Normally switched on

Switched off when

Active Repressor binds to the operator.

This blocks RNA Polymerasefrom transcribing

the genes

I. Bacterial Regulation2. The Basic ConceptOperon

d. Mechanism? (Example – Trp operon)Repressors are specific to their operonSource of Repressor?

Coded for by a Regulatory GeneLocated a distance awayHas its own promotor

2. The Basic ConceptOperon

d. Mechanism? (Example – Trp operon)Repressor Activity

Regulatory genes continually expressedA Few repressors always

presentBinding to operator is reversible

On or off depends number of active repressors around

Repressor – allosteric proteinActive state and inactive stateCorepressor must bind to it

to activate it

Corepressor can act as feedback controlExample – Trp Operon

As tryptophan is produced to high levelsIt binds to repressors activating them

This blocks transcription, shutting downtryptophan production


D. Negative Gene Regulation

Involves a Repressor attaching to the gene operator

Two types 1. Repressible operons

2. Inducible operons


D. Negative Gene Regulation1. Repressible Operons (anabolic)

On = Normal Turn it offNo CoRepessor + CoRepressorRepressor inactive Repr. ActivatedOperon NOT repressed Operon is repress.Protein Synthesis No synthesis

Best Example; The Trp Operon


D. Negative Gene Regulation2. Inducible Operons

(Catabolic)Normally Off Turn it onNo Inducer Make InducerRepressor active Shuts off RepressorOperon blocked Operon activeNo synthesis Synthesis

Best Example;

LAC Operon


E. Positive Gene Regulation

Involves an Activator attaching to genome (CAP for Glucose example)


E. Positive Gene Regulation

Low Glucose High GlucosecAMP up cAMP downBinds to CAP Act. CAP act. LoosesCAP activated CAP inactiveBinds above promotor No synthesis

Synthesis occurs

II. Eukaryotic Regulation

A. Gene regulation especially important for multicellular organisms

Cell specialization

What causes differences?

Differential gene expression!


B. Differential Gene ExpressionExpression of different genes by cells with the same genomeTypical cell – 20% expression

Less in more specialized cellsHow expressed?

Transcription


B. Regulation in Chromatin Structure


C. Regulation by Chromatin Structure

1. Location of a Gene’s promotor

relative to

nucleosomes

scaffold attachment

nuclear lamina

II. Eukaryotic RegulationC. Regulation by Chromatin Structure

2. Location in Heterochromatin?

Highly condensed

Usually not expressed


C. Regulation by Chromatin Structure

3. Chemical modifications to histones and histone tails


3. Chemical modificationsHistone Acetylation

Acetyl groups attached

to lysines in thehistone tails

Effects+ charges neutralizedNo longer bind to

other nucleosomes Stays loosely packed

Deacetylation - repacks


3. Chemical modifications to histones and histone tails

Methyl Groups – promotes condensing

Phosphate Groups – reverses methyl effect

Histone Code Hypothesis

Combinations of

chem modifications are

the key

II. Eukaryotic RegulationD. DNA Methylation

Direct methylation of DNA Bases(usually cytosine)

1. More methylation – DNA less active2. Proteins can bind to methyl groups

Recruit histone deacetylation enzymesResults in a dual mechanism to

repress transcription3. Critical in embryonic development

Cell differentiation Methylation pattern passeddown cell lines

Genomic Imprinting!!


E. Epigenetic Inheritance(Differential expression patterns are passed on)Passing Traits without

code sequence changesThese changes are reversible

F. Regulating Transcription InitiationUltimately – Control the binding and action of RNA Polymerase.1. Basic Organization of a Eukaryotic Gene

Upstream PromotorProteins assemble here =Transcription Init.

ComplexControl Elements

Upstream DNA SequencesBind control proteins

Eukaryotic Gene Organization

F. Regulating Transcription Initiation2. Transcription Factors

RNA Polymerase requires thesea. General transcription factors

required for all protein code genesBind to promotor (TATA) and otherproteins Make

Transcription Initiation complexThen RNA Polymerase can function

Leads to low level transcription

F. Regulating Transcription Initiation

2. Transcription Factors

RNA Polymerase requires these

b. Specific Transcription Factors

Specific to a gene

Interact with control factors

leads to high level transcription

F. Regulating Transcription Initiation3. Control Elements – The basics

a. Proximal Control ElementsClose to Promotor

b. Distal Control ElementsCalled Enhancers1000s of bases away from

promotorA gene may - multiple enhancers

Enhancers specific to a gene


4. Regulation using Control Elements

Key conceptProtein-protein interactions work

to assemble Transcr.Init.Complex on the promotor


4. Regulation using Control Elements

Mechanism

Activators bind to enhancer

Mediator proteins form link betweenactivators and promotor

DNA Bending protein bends

enhancer to promotor

Link builds Transc.Init.Complex

F. Regulating Transcription Initiation4. Regulation using Control Elements

RepressionRepressors (methods)

Bind to enhancers – Blocking

ActivatorsBind to activators – blocking

other protein bindingMany other repression methods

F. Regulating Transcription Initiation5. Enhancer Combination Effects

Enhancer control element sequencesrepeat for many genes

Each enhancer has around 10control element sequences

The combination of the control elements provides specificity for specific genes


6. Coordinate Control of Related genes

Prokaryotes – Operons!

What about Eukaryotes????

a. Related genes may be clustered

close togetherBut – separate promotors


6. Coordinate Control of Related genes

b. Most commonly

Co-expressed genes scattered!

So how?

- Coordination by specific

control element sequences- often response to signal

from

outside of cell – binds to allenhancers with a specific

control element sequence

G. Post-Transcriptional Regulation1. RNA Processing

Alternative RNA Splicing – Different mRNAs

are made from same primary transcript Cell types Regulatory proteins bind to determine intron / exon choices

G. Post-Transcriptional Regulation2. mRNA Degradation

Control of the life span of mRNA3. Initiation of Translation

a. Regulatory proteins attach at 5’ endof mRNAprevents binding of Ribosome

b. Poly-A tail manipulationNecessary for Ribosome bindingSome mRNAs lacking in Poly-ANo translation until enzyme adds

additional As

G. Post-Transcriptional Regulation

2. mRNA Degradation

Control of the life span of mRNA

3. Initiation of Translation

c. manipulation of the proteins thatinitiate translation

G. Post-Transcriptional Regulation4. Protein processing

Control of cleavage and reassembly

Degradation of proteins = limit protein life span

Tag proteins with ubiquitin

Attracts proteasomes to degrade them

III. Impact of Non-Coding RNA

A. Large part of the genome codes for

Non-protein coding RNA (Non-Coding RNA)

Many questions remain on what these do and how many types exist

B. Role in regulation? Two places

1. mRNA translation (C,D, and E)

2. Chromatin configuration (F)

III. Impact of Non-Coding RNAC. Micro-RNA = miRNA

Small and single stranded (ssRNA)

1. Formation – from large precurser RNA

Trimmed by enzyme “Dicer”

miRNA Production

III. Impact of Non-Coding RNAC. Micro-RNA = miRNA

Small and single stranded (ssRNA)

2. Function

Degrades a target mRNA

Blocks translation of a target mRNA

III. Impact of Non-Coding RNAD. siRNA – Small interfering RNA

Turns off genes with same sequence

Made by same machinery that makes miRNA

Slight distinctions – see text pg365

E. Are these informational, catalytic, or structural?

III. Impact of Non-Coding RNAF. Impact on Chromatin Configuration

siRNA – heterochromatin in yeast

Associate with proteins to recruit

enzymes – condensing chromatin

Check experiments – 365-366

IV. Cell DifferentiationA. Key Context – Embryonic Development

B. The Genetic Programming of development

1. Three necessary components

Cell Division

Cell Differentiation

Morphogenesis – Shaping

3D organization of cell types

IV. Cell DifferentiationA. Key Context – Embryonic Development

B. The Genetic Programming of development

2. Key Concepts

Differential gene expression

leads to specialization

Different collections of activators

Different patterns

3. BUT ……

What sets this all up?????

IV. Cell DifferentiationC. Cytoplasmic Determinants and Induction

What tells a cell which collection of genes to express?

Two sources of information

1. The Egg’s cytoplasm

2. Environment around a cell


1. The Egg’s cytoplasm

Not homogeneous

Cytoplasmic Determinants

Maternal substances unevenly

distributed

mitotic divisions – new cells get

differing concentrations


2. Surrounding Environment

Signals from surrounding cells

Contact with surrounding cell surfaces

Growth factors from neighbors

Changes from these sources called

Induction

Cell-surface receptors important

Untilizes signal transduction pathways

IV. Cell DifferentiationD. Sequential Regulation

1. Determination – the events that lead to

observable differentiation of a cell

Completed determination is irreversible

Caused by differential gene expression

for tissue specific proteins


2. Pathway to differentiation

Signal

mRNAs

Specific protein

Observable phenotype

3. Expression pattern is SEQUENTIAL

4. Key point of regulation - transcription


5. There is a hierarchy of differentiation

6. Study muscle cell example – pg368

Cell Differentiation

IV. Cell DifferentiationE. Pattern Formation– the spatial

arrangement of tissues and organs.

Positional Information cues –

establish the three key axes of the body Cytoplasmic determinants

Inductive signals

IV. Cell DifferentiationE. Pattern Formation

1. Information gleaned from

D. melanogaster

IV. Cell Differentiation

E. Pattern Formation


D. melanogaster

a. Modular construction -

ordered series of segments

Head

Thorax

Abdomen

Sub-segments




D. melanogaster

b. 3 Axes

Anterior-posterior

Dorsal- ventral

Laterals (side-side)




D. melanogaster

c. Pattern formation?

Axes from

cytoplasmic determinants



2. Drosophila sequence

Edward B. Lewis (1940s)

Genetic approach to embryology

using D. melanogaster

Born Wilkes-Barre PA

Caltech professor

Nobel prize 1995

Died 2004 (cancer)


Initial discovery by Lewis

Specific gene mutations led to extra legs or wings growing in the wrong places

Homeotic Genes – Control pattern formation

Identification of specifics? 30 years!

Nusskein-Volhard and Wieschaus

(Germany)


Nusskein-Volhard and Wieschaus

(Germany)

Eventually – dicovered 1200 genes

controlling pattern formation

1995 nobel prize for these three!

2. Drosophila sequenceGenes discovered by tracking

recessive mutations

Embryonic Lethals

2. Drosophila sequenceAxis establishment

Maternal Effect Genes – build thecytoplasmic determinants in eggs

Also called Egg-polarity Genes

Example – the Bicoid GeneMutant form leads to

Two tail ends, no headGene sets up the anterior-

posterioraxis


The Bicoid Gene

This gene exemplifies the

Morphogen Gradient Hypothesis

Axes set up by concentration

gradients of morphogens

2. Drosophila sequenceThe Bicoid Gene

Bicoid processBicoid mRNA concentrated in the

anterior end of unfert. EggFertilization = zygoteBicoid mRNA translated to proteinBicoid protein diffuses through

zygoteCreates conc. Gradient

High conc. = anteriorLow conc. = posterior

First gene-protein specifically linked topattern formation


Beyond Bicoid example -

Other gene linked gradients found for

dorsal – ventral axis

Later positional information

Finer scale patterns

correct orientation of each segment

V. Cancer and Gene ExpressionA. Types of Genes associated with Cancer

Deal with cell growth and division

1. Growth factor genes

receptor genes

signaling pathway genes

2. Causes spontaneous mutations

environmental influences

Tumor viruses

V. Cancer and Gene ExpressionA. Types of Genes associated with Cancer

3. Tumor Viruses

First - Payton Rous 1911

Epstein-Barr virus - Mono …

Linked to lymphoma

Papillomaviruses - cervical cancer

HTLV-1 - Leukemia

V. Cancer and Gene ExpressionA. Types of Cancer Genes

4. Oncogenes and Proto-Oncogenes

Proto-Oncogenes - normal versions

Promote growth and division

Oncogene - mutated - leads to cancer


4. Oncogoenes and Proto-Oncogenes

How proto-oncogene to oncogene?

Mutation effects protein product

a. DNA moves in genome

(protoOncogene move to

active promotor)

b. amplification of gene

(increase copies of gene)

c. point mutation


4. Oncogoenes and Proto-Oncogenes

How proto-oncogene to oncogene?

Mutation effects protein product

c. point mutation

In promotor or enhancer

increasing expression

In gene itself

protein more active or

resistant


5. Tumor-Suppressor Genes

Genes that inhibit growth or division

How?

Repair damaged DNA

Control cell adhesion

Cell signaling to control division

Mutations can lead to tumors

V. Cancer and Gene ExpressionB. Interference with Cell-Signaling

Two key genes studied

ras proto-oncogene

p53 tumor suppressor gene

1. Ras gene ----- ras protein (G-protein)

Normal - growth factor

growth factor receptor

G-protein

Kinase cascade

Cell cycle stimulus


1. Ras gene ----- ras protein (G-protein)

Mutated gene

hyperactive ras protein

triggers cascade without signal

excessive cell division


2. P53 gene - activated by DNA damage

encodes transription factor

promotes synthesis of cycle inhibitors

such as p21 - binds cdk’s

activates DNA repair genes

activates Apoptosis genes

p53 = guardian angel of the genome

V. Cancer and Gene ExpressionC. Multistep Model for Cancer

Multiple mutations necessary for cancer

Cancer more common with age.

Example

Colorectal cancer

Needs ras oncogene and

mutated p53 gene

Most cancers need

At least one oncogene

multiple damaged tumor suppressors

V. Cancer and Gene ExpressionC. Multistep Model for Cancer

In many malignant tumors

Telomerase gene is activated!

Increasing life span of tumor

V. Cancer and Gene ExpressionD. Inherited Cancer Predisposition

1. Key - since many genetic problems are

needed to make cancer,

if one is inherited - one step closer!

Best example - BRCA1 and BRCA2

mutations

Increase Breast Cancer risk

Mary-Claire King, 1990

Chapter 18 Regulation of Gene Expression. Genetic Diversity in Asexual Bacteria? I. From Within A....

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