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How Genes Are Regulated
1
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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2
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
Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or displayHartwell et al., 4th edition, Chapter 16
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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
Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or displayHartwell et al., 4th edition, Chapter 16
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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
Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or displayHartwell et al., 4th edition, Chapter 16
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TATAA
TAA
T
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t rans-acting factors interact with c is-actingelements to control transcription initiation
Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or displayHartwell et al., 4th edition, Chapter 16
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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
Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or displayHartwell et al., 4th edition, Chapter 16
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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
Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or displayHartwell et al., 4th edition, Chapter 16
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Basal factors bind to promoters of allprotein-encoding genes
Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or displayHartwell et al., 4th edition, Chapter 16
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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
Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or displayHartwell et al., 4th edition, Chapter 16
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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
Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or displayHartwell et al., 4th edition, Chapter 16
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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
Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or displayHartwell et al., 4th edition, Chapter 16
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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
Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or displayHartwell et al., 4th edition, Chapter 16
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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
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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
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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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