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Chapter 17Chapter 17Control of Gene Regulation inControl of Gene Regulation in

EukaryotesEukaryotes

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Eukaryotic Gene Regulation DNA binding proteins also play important

roles in regulating gene expression in

eukaryotic cells

However, there are several important

differences

Eukaryotic genes are not organized inoperons and transcribed together as a

single mRNA

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Each structural gene has its own promoter and

is transcribed independently

Monocistronic mRNA - each mRNA codes

for a single polypeptide

Polycistronic mRNA - the mRNA codes for

two or more polypeptides are only found in

prokaryotes

Chromatin structure affects gene expression Transcription requires unwinding of the DNA

from the histone proteins

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Eukaryotic cells have many moretranscriptional activators as well as

repressors

Again, transcription and translation are

physically separated so the regulatory

mechanisms in eukaryotic cells mustdiffer from those in bacteria

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Chromatin Structure andGene Regulation

DNA tightly wound around nucleosomes

tends to repress transcription

Efficient transcription requires changes

in chromatin structure to make the DNA

more accessible to transcription factors,

activator proteins and RNA polymerase

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DNAse I Hypersensitivity Use a nuclease as a tool to probe chromatin

structure

Rationale: DNA tightly wound around nucleosomeswill be resistant to the nuclease since the DNA is

protected by the histone proteins

Changes in chromatin that make the DNA less

tightly associated with the nucleosomes will be seen

as an increased sensitivity to degradation by a

nuclease

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DNAse I Hypersensitivity The DNA located ~1,000 bp upstream of the

promoter of transcriptionally active genes is

hypersensitive to cleavage by DNAse I

Indicates that the chromatin has become more

relaxed and the DNA is more loosely associated

with the nucleosomes

Regulatory proteins and RNA polymerase have

better access to the gene promoter

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Histone Acetylation Nucleosomes are composed of eight histoneproteins Histones have positively charged tails that

interact with the negatively-charged phosphates of

the DNA

Acetyl group can be transferred to the lysine and

arginine amino acids that have the positive charge

by acetyl transferases

Neutralizes the positive charge so the DNA is

more loosely associated with the histoneAcetyl group

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Flowering in Arabidopsis

Arabidopsis is an important genetic modelfor plant systems

Text describes an example of controllingexpression of genes involved with floweringthat involves acetylation and deactylationand its effect on transcription

Read this section for a better understandingof this regulatory process (see Figure 17.3).

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DNA Methylation Methylation of cytosine at position 5 is

another important mechanism for affecting

transcription

Heavily methylated DNA is associated with

repression of transcription

Unmethylated DNA is associated with DNA

that is available for transcription

Usually occurs in CpG islands

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CpG Islands Simply means that a methylated C is usuallyfollowed by a G

The p represents the phosphodiester bond Inactive genes have methylated CpG

Find heavy methylation of CpG with fully

repressed genes such as those on theinactive X-chromosome of mammalian

females

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Chromatin Remodeling Some transcription factors or other

regulatory proteins alter chromatin

structure without affecting histonesdirectly

For example, nucleosomes can be

repositioned to allow binding andinitiation of transcription

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Epigenetics Changes to DNA and chromatin that affect traits that

are passed on to other cells or even future generations

But not caused by changes in DNA sequence

Example - maternal behavior

The offspring of rats where the mother licks and

grooms them more show less fearful and stress-related responses as adults

Additional examples in text

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Changes in chromatin must be stable through chromosomereplication if the epigenetic effect is inheritable

Key mechanism appears to be DNA methylation

CpG methylation means there are methylated cytosine on

opposite strands

After replication, only the old strand is methylated -

hemimethylated

Methyltransferase methylates adds -CH3 tounmethylatednew strand

siRNA, miRNA and piRNA also play a role in some cases

Molecular Mechanism

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Transcriptional Control in

Eukaryotic Cells Review from Chapter 13

General transcription factors assemble into a basal

transcription apparatus

Binds to the core promoter that is located

immediately upstream of the gene

Transcriptional activator proteins enhance the rate oftranscription by stimulating (or stabilizing) the basal

transcription apparatus

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Transcriptional Activator

Proteins Two distinct functions

One domain of the proteins binds to a specific DNA

sequence

Another domain can interact with a different

component of the transcriptional apparatus to enhance

the rate of transcription

A coactivatorcan serve to link the transcription factor

to another component of the transcription machinery

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Transcriptional activators usually bindto either:

A regulatory promoter that isupstream of the core promoter

An enhancer, which may be located

several thousand bases from thepromoter

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GAL4 from Yeast GAL4 is a transcriptional activator that

regulates transcription of several yeast genes

involved in galactose metabolism

Widely used in molecular biology as a research

tool since most other eukaryotes do not have a

homologous transcription factor

Well-studied example of a transcriptional

activator

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The DNA binding site for GAL4 protein is

called UASG forupstream activator

sequence forGAL4

Binding of GAL4 to UASG activates

transcription

GAL4 activity is regulated by GAL80

In the absence of galactose, GAL80binds to GAL4, which prevents binding

to UASG and no transcription

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In the presence of galactose, GAL80binds galactose

Allosteric change prevents binding ofthe GAL80-galactose complex to

GAL4

GAL4 is free to bind to the UASG andtranscription initiates

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No galactose

+ galactose

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Enhancers and Insulators Enhancers can affect transcription even

when they are located at a considerable

distance from the target gene

In general, activator protein binds to the

enhancer

The enhancer loops out to bring theactivator protein closer to the basal

transcription apparatus

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Insulators

Most enhancers could affect any

gene within their proximity

Insulators (also called boundary

elements) are DNA sequences that

function to limit (or isolate) theactivity of an enhancer

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Coordinated Gene Regulation Some genes need to be expressed in a

coordinated manner, but eukaryotic genes arenot organized in operons

Heat shock genes are one example

Thermal stress (and other types of stress)leads to increased transcription of ~20

different genes How are the 20 genes coordinately expressed

when they dispersed throughout the genome?

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Response ElementsEach heat shock gene shares a

common regulatory DNA sequence

called a response element

Usually have a short consensus

sequence

Gene may have additional response

elements

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One gene can be activated by a variety of stimuli by

have several different response elements

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Common Feature of Eukaryotic

Transcriptional Control

A single gene can be activated by several

different response elements

Multiple response elements allow a single gene to

be activated by different stimuli

Presence of the same response element ondifferent genes allows coordinated expression of a

set of genes in response to a common stimulus

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mRNA ProcessingAlternative splicing of exons of the pre-mRNA

can yield different proteins

Use an example from Drosophila

Sxl(sex lethal) protein is synthesized in female

cells

Sxlis an RNA binding protein and binds to the

tra (transformer) pre-mRNA and affects splicing

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In the absence of sxl protein (males), splicing of the

tra mRNA occurs at a different place

In females, the sxl protein binds to the pre-mRNA to

prevent splicing

Splicing at an alternative site

Result is mRNA in males codes for an inactive tra

protein

The alternative splicing in females leads to a tra

mRNA that codes for a functional protein

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Also seeFigure

17.11

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RNA Stability Eukaryotic mRNA is, in general, more long livedthan prokaryotic mRNA

Some eukaryotic mRNAs are stable for hours,

days or even months

Can results in very large differences in the

amount of protein made

Degradation usually begins at the cap structure

on the 5 end

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RNA SilencingA recently discovered mechanism for

silencing gene expression by targeted

degradation of the mRNA

Possibly as many as 30% of genes utilize

RNA silencing

Noble prize awarded in 2006

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Translational and

PosttranslationalC

ontrol There are factors that can bind the mRNA in the 5UTR, which affects the rate of translation

Although rarer, some factors bind to 3 UTR to

affect translation

Many proteins are initially made in an inactive state

Activation requires addition processes suchremoval of some amino acids, as well as chemical

modification (methylation, acetylation,

phosphorylation, etc.)