1. Eukaryotic Gene Regulation

8/13/2019 1. Eukaryotic Gene Regulation

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How Genes Are Regulated

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PART II

Gene Regulation inEukaryotes

2.1 Overview of Eukaryotic Gene Regulation

2.2 Control of Transcription Initiation

2.3 Epigenetic Effects

2.4 Regulation After Transcription (siRNA and miRNA)

OUTLINE


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The focus of this course is on human genetics

Genetics has powerful tools for understanding humanbiology

Paradigm shift from studying one gene or protein at a time

to studying interacting networks of many genes andproteins

Molecular studies can lead to predictive and preventivemedicine

DNA diagnostics can be used to generate a geneticprofile of an individual

Design of therapeutic drugs to prevent or minimizesymptoms of gene-based diseases


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Modern genetic techniques

Genetic dissection of model organisms

Inactivate a gene and observe the consequences

Genome sequencing

Human Genome Project

Model organisms and other organisms

Understanding higher-order processes that arise frominteracting biological networks

Genomics can rapidly analyze thousands of genes

High-throughput DNA sequencing and genotyping

Large-scale DNA arrays (chips)


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The Human Genome Project

First formally discussed in 1985

Officially began in 1990 estimated 15 years, $3 billion

Systems approach to biology and medicine

Study of the interplay of the elements in a biologicalsystem as it undergoes genetic perturbation or biologicalactivation

Development of technologies to analyze the genome andthe complete biochemical machinery of cells

3-5% of budget committed to studying the ethical, legal,and social implications (ELSI) of human genome mapping

Social and personal repercussions are generating newareas of biological concern

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Global analysis of genes and their mRNAs

Genomics

Studies whole genomes: global analysis of chromosomalfeatures and gene products

the development and application of more effective mapping,sequencing, and computational tools

Predict and verify the existence and functions of previouslyundefined genes using molecular biology tools

High throughput instruments - DNA sequencers and DNAarrays

Platformsall components needed for automatedacquisition of data

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Important implications of genetics tosocial issues

Entire genetic profiles of individuals will become available

This genetic information can be used to help people

Make predictions about future possibilities and risks

Or, genetic information could also be used to restrictpeople's lives

Genetic Information Nondiscrimination Act was passed

by the US federal government in 2008

Prohibits discrimination on the basis of genetic tests byinsurance companies and employers


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Important implications of genetics tosocial issues (continued)

Proper interpretation of genetic information andunderstanding of statistical concepts is essential

Regulation and control of new technology

Transgenic technology (genetic engineering) is routinein many animals

Should genetic engineering of human embryos beallowed?

Guidelines must be established to prevent misuse of newknowledge in human genetics


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Overview of eukaryotic gene regulation

Eukaryotes use complex sets of interactions

Regulated interactions of large networks of genes

Each gene has multiple points of regulation

transcription takes place in the nucleus and translation takesplace in the cytoplasm

Genes are turned on or off in the right place and

time

Differentiation and precise positioning of tissuesand organs during embryonic development

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Key regulatory events in eukaryotes

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Multiple steps whereproduction of the

final gene productcan be regulated in

eukaryotes

Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or displayHartwell et al., 4th edition, Chapter 16

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Control of transcription initiation

Three types of RNA polymerases in eukaryotes

RNA pol I transcribes rRNA genes

RNA pol II

transcribes all protein-coding genes(mRNAs) and micro-RNAs

RNA pol III transcribes tRNA genes and some smallregulatory RNAs


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RNA polymerase II transcription

RNA pol II catalyzes synthesis of the primary transcript,which is complementary to the template strand of the gene

Most RNA pol II transcripts undergo further processing togenerate mature mRNA

RNA splicing removes introns

Addition of 5' GTP cap protects RNA fromdegradation

Cleavage of 3' end and addition of 3' polyA tail


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c is-acting elements: promoters and enhancers

Promoters

usually directly adjacent to the gene

Include transcription initiation site

Often have TATA box:

Allow basal level of transcription

Enhancerscan be far away from gene

Augment or repress the basal level of transcription


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TATAA

TAA

T


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t rans-acting factors interact with c is-actingelements to control transcription initiation


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Direct effects oftranscription factors:

Through binding toDNA

Indirect effect oftranscription factors:

Through protein-

proteininteractions


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Use of reporter genes to identify t rans-actingfactors in transcriptional regulation


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Basal transcription factors

Basal transcription factors assist the binding of RNA pol IIto promoters

Key components of basal factor complex:

TATA box-binding protein (TBP) Bind to TATA box

First of several proteins to assemble at promoter

TBP-associated factors (TAFs)

Bind to TBP assembled at TATA box

RNA pol II associates with basal complex and initiates basallevel of transcription


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Basal factors bind to promoters of allprotein-encoding genes


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Ordered pathway ofassembly at promoter:

1. TBP binds to TATA box

2. TAFs bind to TBP

3. RNA pol II binds to TAFs


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Binding of activators to enhancersincreases transcriptional levels

Low level transcription occurswhen only basal factors arebound to promoter


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When basal factors andactivators are bound to DNA,

rate of transcription increases


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Domains within activators

Activator proteins have two functional domains

Sequence-specific DNA binding domain

Binds to enhancer

Transcription-activator domain

Interacts with other transcriptional regulatory proteins

Some activators have a third domain

Responds to environmental signals

Example - steroid hormone receptors


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DNA-binding domains of activator proteins

Interacts with major grooveof DNA

Specific amino acids havehigh-affinity binding to

specific nucleotide sequenceThe three best-characterizedmotifs:

Helix-loop-helix (HLH)

Helix-turn-helix (HTH)

Zinc finger


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Repressor proteins suppress transcriptioninitiation through different mechanisms

Some repressors have no effect on basal transcription butsuppress the action of activators

Compete with activator for the same enhancer

OR

Block access of activator to an enhancer

Some repressors eliminate virtually all basal transcriptionfrom a promoter

Block RNA pol II access to promoter

OR

Bind to sequences close to promoter or distant frompromoter


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Complex regulatory regions enablefine-tuning of gene expression

Each gene can have many regulatory proteins

In humans, ~2000 genes encode transcriptionalregulatory proteins

Each regulatory protein can act on many genes

Each regulatory region can have dozens of enhancers

Enhanceosome multimeric complex of proteins and othersmall molecules that associate with an enhancer

Enhancers can be bound by activators and repressorswith varying affinities

Different sets of cofactors and corepressors competefor binding to activators and repressors


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Chromatin structure and epigenetic effects

Chromatin structure can affect transcription

Nucleosomes can sequester promoters and make theminaccessible to RNA polymerase and transcriptionfactors

Histone modification and DNA methylation

Chromatin remodeling and hypercondensation

Epigenetic changes

changes in chromatin structure thatare inherited from one generation to the next

DNA sequence is not altered


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Chromatin reduces transcription


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Genomic imprinting results fromtranscriptional silencing

Genomic imprinting

expression of a gene depends onwhether it was inherited from the mother or father

Occurs with some genes of mammals

Epigenetic effect (no change in DNA sequence)

Paternally imprinted gene is transcriptionally silenced if itwas transmitted from the father

Maternal allele is expressed

Maternally imprinted gene is transcriptionally silenced if itwas transmitted from the mother

Paternal allele is expressed


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Methylation of complementary strandsof DNA in genomic imprinting

Epigenetic state can bemaintained across cellgenerations through theaction of DNA methylases


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The resetting ofgenomic imprints

during meiosis

Epigenetic imprints remainthroughout the lifespan of themammal

In germ cells, epigeneticimprints are reset at eachgeneration

During meiosis, imprints are

erased and new ones are set


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Genomic imprinting and human disease

Examples: two syndromes associated with small deletionsin chromosome 15

At least two genes within this region are differentlyimprinted

Praeder-Willi syndrome occurs when the deletion isinherited from the father

Angelman syndrome occurs when the deletion isinherited from the mother

Affected individuals have mental retardation anddevelopment disorders


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Inheritance patterns of disorders resultingfrom mutations in imprinted genes

These pedigrees may appear to be instances of incompletepenetrance, but are distinctly different


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Regulation after transcription

Posttranscriptional regulation can occur at any step

At the level of RNA

Splicing, stability, and localization

Example alternative splicing of mRNA

Generates more diversity of proteins

Common feature in eukaryotes

At the level of protein

Synthesis, stability, and localization


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Some small RNAs are responsible forRNA interference (RNAi)

Specialized RNAs that prevent expression of specific genesthrough complementary base pairing

Small (21 30 nt) RNAs

Micro-RNAs (miRNAs) and small interfering RNAs

(siRNAs)

First miRNAs (l in-4and let-7) identified in C. elegans

Nobel prize to A. Fire and C. Mello in 2006

Posttranscriptional mechanisms for gene regulation mRNA stability and translation

May also affect chromatin structure


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Two ways that miRNAs can down-regulateexpression of target genes

When complementarity isperfect:

Target mRNA is degraded


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When complementarity is imperfect:

Translation of mRNA target isrepressed


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siRNAs detect and destroy foreign dsRNAs

Two biological sources of dsRNAs that are precursors ofsiRNAs (pri-RNAs)

Transcription of both strands of an endogenousgenomic sequence

Arise from exogenous virus

pri-RNAs are processed by Dicer

siRNA pathway targets dsRNAs for degradation

siRNAs are very useful experimental tools to selectivelyknock down expression of target genes

To study function of a gene, dsRNAs for that gene canbe introduced into cells


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The cellularcomponents of

gene expression

Mutations in genesencoding gene productsfor transcription, RNA

processing, translation,and protein processing areoften lethal

Some mutations in tRNA

genes can suppressmutations in protein-coding genes

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Impact of unrepaired mutations

Germ line mutations occur in gametes or in gameteprecursor cells

Transmitted to next generation

Provide raw material for natural selection

Somatic mutations occur in non-germ cells

Not transmitted to next generation of individuals

Can affect survival of an individual

Can lead to cancer

Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or displayHartwell et al., 4th ed., Chapter 7

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Alkaptonuria: An inborn error of metabolism


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The molecularbasis of sickle-cell

anemia

GluVal substitution atsixth amino acid affectsthe three-dimensionalstructure of the

hemoglobin chain

Abnormal proteinaggregates cause sickle

shape of red blood cells

Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or displayHartwell et al., 4th ed., Chapter 7 44


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A comprehensive example:Mutations that affect vision


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The cellular basis of vision The molecular basis of vision


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How mutations modulate light and colorperception


Autosomal

dominantdisorder


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Levels of polypeptide structure

Interactions that determine thethree-dimensional conformationof a polypeptide



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Levels of polypeptide structure (cont)


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1ostructure is the amino acid sequence

2ostructure is the characteristic geometry of localizedregions


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Levels of polypeptide structure (cont)


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3ostructure is the completethree-dimensionalarrangement of a polypeptide


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Multimeric proteins are complexes ofpolypeptide subunits


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Identical subunits Non-identical subunits


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Multimeric proteins are complexes ofpolypeptide subunits (cont)

One polypeptide in different proteins

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1. Eukaryotic Gene Regulation