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PART II

How Genes Are Regulated

Gene Regulation in Eukaryotes

OUTLINE
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)

The focus of this course is on human genetics

Genetics has powerful tools for understanding human biology


Paradigm shift from studying one gene or protein at a time to studying interacting networks of many genes and proteins Molecular studies can lead to predictive and preventive medicine DNA diagnostics can be used to generate a genetic profile of an individual Design of therapeutic drugs to prevent or minimize symptoms 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 from interacting biological networks
Genomics can rapidly analyze thousands of genes High-throughput DNA sequencing and genotyping Large-scale DNA arrays (chips)

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 biological system as it undergoes genetic perturbation or biological activation Development of technologies to analyze the genome and the 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 new areas of biological concern
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Global analysis of genes and their mRNAs


Genomics

Studies whole genomes: global analysis of chromosomal


features and gene products

the development and application of more effective mapping,


sequencing, and computational tools

Predict and verify the existence and functions of previously


undefined genes using molecular biology tools

High throughput instruments - DNA sequencers and DNA arrays Platforms all components needed for automated acquisition of data
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Important implications of genetics to social 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 restrict people's lives Genetic Information Nondiscrimination Act was passed by the US federal government in 2008
Prohibits discrimination on the basis of genetic tests by insurance companies and employers

Important implications of genetics to social issues (continued)


Proper interpretation of genetic information and understanding of statistical concepts is essential

Regulation and control of new technology


Transgenic technology (genetic engineering) is routine in many animals Should genetic engineering of human embryos be allowed? Guidelines must be established to prevent misuse of new knowledge in human genetics

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 takes place in the cytoplasm

Genes are turned on or off in the right place and time Differentiation and precise positioning of tissues and organs during embryonic development
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Key regulatory events in eukaryotes

Multiple steps where production of the final gene product can be regulated in eukaryotes

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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 small regulatory 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 to generate mature mRNA

RNA splicing removes introns


Addition of 5' GTP cap protects RNA from degradation Cleavage of 3' end and addition of 3' polyA tail

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cis-acting elements: promoters and enhancers


Promoters usually directly adjacent to the gene

Include transcription initiation site

A A A T T Allow basal level of transcription


Often have TATA box: TATA Enhancers can be far away from gene Augment or repress the basal level of transcription

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trans-acting factors interact with cis-acting elements to control transcription initiation


Direct effects of transcription factors:

Through binding to DNA


Indirect effect of transcription factors: Through proteinprotein interactions

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Use of reporter genes to identify trans-acting factors in transcriptional regulation

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Basal transcription factors


Basal transcription factors assist the binding of RNA pol II to 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 basal level of transcription
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Basal factors bind to promoters of all protein-encoding genes


Ordered pathway of assembly at promoter: 1. TBP binds to TATA box

2. TAFs bind to TBP


3. RNA pol II binds to TAFs

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Activators are transcription factors that bind to enhancers


Activators are responsible for much of the variation in levels of transcription of different genes Increase levels of transcription by interacting directly or indirectly with basal factors at the promoter 3-dimensional complex of proteins and DNA Mechanisms of activator effects on transcription Stimulate recruitment of basal factors and RNA pol II to promoters Stimulate activity of basal factors already assembled on promoters Facilitate changes in chromatin structure
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Binding of activators to enhancers increases transcriptional levels


Low level transcription occurs when only basal factors are bound to promoter

When basal factors and activators 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 groove of DNA Specific amino acids have high-affinity binding to specific nucleotide sequence

The three best-characterized motifs:


Helix-loop-helix (HLH)

Helix-turn-helix (HTH)
Zinc finger
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Repressor proteins suppress transcription initiation through different mechanisms


Some repressors have no effect on basal transcription but suppress 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 transcription from a promoter Block RNA pol II access to promoter OR Bind to sequences close to promoter or distant from promoter
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Repressor proteins that act through competition with an activator protein


Repressor binds to the same enhancer sequence as the activator Has no effect on the basal transcription level

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Repressor proteins that act through quenching an activator protein


Quenchers bind to the activator but do not bind to DNA
Type I: Repressor blocks the DNA-binding domain

Type II: Repressor blocks the activation domain

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Complex regulatory regions enable fine-tuning of gene expression


Each gene can have many regulatory proteins

In humans, ~2000 genes encode transcriptional regulatory proteins


Each regulatory protein can act on many genes

Each regulatory region can have dozens of enhancers


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

Enhancers can be bound by activators and repressors with varying affinities


Different sets of cofactors and corepressors compete for 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 them inaccessible to RNA polymerase and transcription factors
Histone modification and DNA methylation Chromatin remodeling and hypercondensation Epigenetic changes changes in chromatin structure that are 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 from transcriptional silencing


Genomic imprinting expression of a gene depends on whether 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 it was transmitted from the father
Maternal allele is expressed

Maternally imprinted gene is transcriptionally silenced if it was transmitted from the mother
Paternal allele is expressed
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Methylation of complementary strands of DNA in genomic imprinting


Epigenetic state can be maintained across cell generations through the action of DNA methylases

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The resetting of genomic imprints during meiosis


Epigenetic imprints remain throughout the lifespan of the mammal In germ cells, epigenetic imprints are reset at each generation 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 deletions in chromosome 15 At least two genes within this region are differently imprinted Praeder-Willi syndrome occurs when the deletion is inherited from the father Angelman syndrome occurs when the deletion is inherited from the mother

Affected individuals have mental retardation and development disorders

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Inheritance patterns of disorders resulting from mutations in imprinted genes


These pedigrees may appear to be instances of incomplete penetrance, 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 for RNA interference (RNAi)


Specialized RNAs that prevent expression of specific genes through complementary base pairing Small (21 30 nt) RNAs Micro-RNAs (miRNAs) and small interfering RNAs (siRNAs)
First miRNAs (lin-4 and 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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Primary transcripts containing miRNA


Most miRNAs are transcribed by RNA polymerase II from noncoding DNA regions that generate short dsRNA hairpins

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miRNA processing
Drosha excises stem-loop from primary miRNA (pri-miRNA) to generate pre-miRNA of ~ 70 nt Dicer processes pre-miRNA to a mature duplex miRNA One strand is incorporated into miRNA-induced silencing complex (RISC)

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Two ways that miRNAs can down-regulate expression of target genes


When complementarity is perfect: When complementarity is imperfect:

Target mRNA is degraded

Translation of mRNA target is


repressed

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siRNAs detect and destroy foreign dsRNAs


Two biological sources of dsRNAs that are precursors of siRNAs (pri-RNAs) Transcription of both strands of an endogenous genomic sequence Arise from exogenous virus pri-RNAs are processed by Dicer siRNA pathway targets dsRNAs for degradation siRNAs are very useful experimental tools to selectively knock down expression of target genes To study function of a gene, dsRNAs for that gene can be introduced into cells
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siRNA video

http://www.youtube.com/watch?v=Fa4skYBJHoI

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The cellular components of gene expression


Mutations in genes encoding gene products for transcription, RNA processing, translation, and protein processing are often lethal Some mutations in tRNA genes can suppress mutations in proteincoding genes

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Health consequences of mutations in genes encoding DNA repair enzymes

Xeroderma pigmentosum: Mutations in one of seven genes encoding enzymes involved in nucleotide excision repair

Hereditary forms of colorectal cancer (not shown): Mutations in human homologs of bacterial genes (MutS and MutL) involved in mismatch repair
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Impact of unrepaired mutations


Germ line mutations occur in gametes or in gamete precursor 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
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Alkaptonuria: An inborn error of metabolism

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The molecular basis of sickle-cell anemia


GluVal substitution at sixth amino acid affects the three-dimensional structure of the hemoglobin b chain

Abnormal protein aggregates cause sickle shape of red blood cells

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Sickle-cell anemia is pleiotropic

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A comprehensive example: Mutations that affect vision


The cellular basis of vision The molecular basis of vision

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How mutations modulate light and color perception

Autosomal dominant disorder

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Levels of polypeptide structure

Interactions that determine the three-dimensional conformation of a polypeptide

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Levels of polypeptide structure (cont)


1o structure is the amino acid sequence

2o structure is the characteristic geometry of localized regions

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Levels of polypeptide structure (cont)

3o structure is the complete three-dimensional arrangement of a polypeptide

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Multimeric proteins are complexes of polypeptide subunits


Identical subunits Non-identical subunits

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Multimeric proteins are complexes of polypeptide subunits (cont)


One polypeptide in different proteins

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