9 - Gene Expression 3 - Regulation

8/8/2019 9 - Gene Expression 3 - Regulation

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REGULATION OFREGULATION OFGENE EXPRESSIONGENE EXPRESSION

Paul D. Brown, PhD

FMS, Basic Medical Sciences(Biochemistry)

[email protected]

Room 6 Biochemistry spine


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Learning Objectives

Describe the mechanisms of gene

regulation in prokaryotic andeukaryotic cells

Identify strategies for measuring

gene expression


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The Paradigm of GeneExpression

Quantitative and qualitative variationsin mRNAs and proteins in cells

mRNA/protein complexity a functionof environmental conditions, stressand development

mRNA/protein complexity a functionof different types of cells in metazoa


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The Paradigm of GeneExpression

Not all genes expressed at onetime from prokaryotic or complex

eukaryotic genomeTypical constancy of cellular DNA

Therefore, differential gene

expressionHow does this occur?

Transcriptional regulation (Primary)

Post-transcriptional regulation


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Gene Regulation

Making gene products or actionsMaking gene products or actionsavailable at appropriate points inavailable at appropriate points indevelopment, or under appropriatedevelopment, or under appropriatecircumstances in the life of ancircumstances in the life of anorganismorganism

Coupled eventsCoupled events


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Jenner & Young.Nature Rev.Microbiol. 2005;

3:281-294

A Common Host-TranscriptionalResponse


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What allows some strains ofSalmonellato be host specific while others are

permitted general admission?


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Strategies for controlling

prokaryotic gene expression

Primarily Transcriptional

Positive regulationNegative regulation

Catabolite repression

Substrate induction

Attenuation


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Gene Control in Prokaryotes

In bacteria, genes clustered intooperonsoperons

OperonOperon = Gene cluster thatencode proteins necessary forcoordinated function


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Operon servesto facilitate

transcriptionalandtranslational

events


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RNA is polycistronic (multiple proteins)Two major modes of transcriptional

regulation

Induction (e.g.,

lacoperon)

In presence of lactose, operon isswitched ON and lactose is metabolized;If glucose is also present, Catabolite-repression occurs, and glucose ismetabolized preferentially

Repression (e.g., trp operon)

In presence of high [Trp], Trp binds to

repressor protein (co-repressor), whichswitches the o eron OFF


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Some Global Events: Prokaryotes

Production of heat-shock (andrelated) proteins

May enhance survival in harshconditions

Basis for pathogenesis

Endospore formation

Suspended vegetative growth to ensuresurvival until conditions are favourable


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Like unicellular organisms, the tens ofthousands of genes in the cells ofmulticellular eukaryotes are continually

turned on and off in response to signalsfrom their internal and externalenvironments.

Gene expression must be controlled on a

long-term basis during cellulardifferentiation, the divergence in formand function as cells specialize. Highly specialized cells, like nerves or

muscles, express only a tiny fraction of theirgenes.

Gene Control in Eukaryotes


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Problems with gene expression and controlcan lead to imbalance and diseases,including cancers.

Controls of gene activity in eukaryotes

involves some of the principles describedfor prokaryotes. The expression of specific genes is commonly

regulated at the transcription level by DNA-binding proteins that interact with otherproteins and with external signals.

With their greater complexity, eukaryotes haveopportunities for controlling gene expression atadditional stages.

Gene Control in Eukaryotes


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Each stage in the entire process ofgene expression provides a potential

control point where gene expressioncan be turned on or off, speeded up orslowed down.

A web of control connects differentgenes and their products.

Gene Control in Eukayotes


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Chromatinpacking

Transcription

RNA processing

Translation

Post-translationalmodification

Levels of Control


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In addition to its role in packing DNAinside the nucleus, chromatinorganization impacts regulation.

Genes of densely condensedheterochromatin are usually notexpressed, presumably becausetranscription proteins cannot reach the

DNA.A genes location relative to nucleosomesand to attachments sites to thechromosome scaffold or nuclear laminacan affect transcription.

Chromatin modifications affect theavailability of genes for transcription


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DNA methylation

Inactive DNA is generally highly methylatedcompared to DNA that is actively transcribed.

For example, the inactivated mammalian Xchromosome in females is heavily methylated.

Genes are usually more heavily methylated in cellswhere they are not expressed.

Demethylating certain inactive genes turns them on. Methylation pattern accounts for genomic

imprinting in which methylation turns offeither the maternal or paternal alleles of certaingenes at the start of development.

Chemical modifications of chromatinplay a key role in chromatin structure

and transcription regulation


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Histone acetylation and deacetylation appearto play a direct role in the regulation of genetranscription.

Acetylated histones grip DNA less tightly, providingeasier access for transcription proteins in this region.

Some of the enzymes responsible for acetylation ordeacetylation are associated with or are componentsof transcription factors that bind to promoters.

In addition, some DNA methylation proteins recruithistone deacetylation enzymes, providing amechanism by which DNA methylation and histonedeacetylation cooperate to repress transcription.

Chromatin modifications affect theavailability of genes for transcription


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Chromatin-modifying enzymes provide acoarse adjustment to gene expression by

making a region of DNA either moreavailable or less available for transcription.

Fine-tuning begins with the interaction oftranscription factors with DNA sequences

that control specific genes. Initiation of transcription is the most

important and universally used controlpoint in gene expression.

Transcription initiation is controlled byproteins that interact with DNA and with

each other


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each other


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Eukaryotic RNA polymerase is dependent ontranscription factors before transcriptionbegins. One transcription factor recognizes the TATA box.

Others in the initiation complex are involved inprotein-protein interactions.

High transcription levels require additionaltranscription factors binding to other control

elements. Distant control elements, enhancers, may be

thousands of nucleotides away from thepromoter or even downstream of the gene or

within an intron.


each other


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each other


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each other


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In prokaryotes, coordinately controlledgenes are often clustered into an operonwith a single promoter and other controlelements upstream. The genes of the operon are transcribed into a

single mRNA and translated together

In contrast, only rarely are eukaryoticgenes organized this way.

Coordinated gene expression in eukaryotesprobably depends on the association of aspecific control element or collection ofcontrol elements with every gene of a

dispersed group.

Gene Regulation: Prokaryote vsEukaryote


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Gene expression may be blocked orstimulated by any post-transcriptional

step.

By using regulatory mechanisms thatoperate after transcription, a cell can

rapidly fine-tune gene expression inresponse to environmental changeswithout altering its transcriptional

patterns.

Post-transcriptional mechanisms playsupporting roles in the control of

gene expression


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RNA processing in the nucleus andthe export of mRNA to the cytoplasmprovide opportunities for gene

regulation that are not available inbacteria.

Possibility of

alternativeRNA splicing

Post-transcriptional mechanisms


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mRNA life span is an importantfactor determining the pattern ofprotein synthesis.

Prokaryotic mRNA molecules may bedegraded after only a few minutes.

Eukaryotic mRNAs endure typicallyfor hours or even days or weeks.For example, in red blood cells the

mRNAs for the hemoglobin polypeptidesare unusually stable and are translatedrepeatedly in these cells.



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Enzymatic shortening of thepoly(A) tailTriggers the enzymatic removal of the 5

cap.Followed by rapid degradation of the

mRNA by nucleases.

Nucleotide sequences in the

untranslated trailer region affectmRNA stability.Transferring such a sequence from a

short-lived mRNA to a stable mRNA

results in quick mRNA degradation.



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Translation of specific mRNAs can beblocked by regulatory proteins that bindto specific sequences or structures withinthe 5 leader region of mRNA.

This prevents attachment to ribosomes. Protein factors required to initiate

translation in eukaryotes offer targets forsimultaneously controlling translation ofall

the mRNA in a cell. This allows the cell to shut down translation if

environmental conditions are poor (for example,shortage of a key constituent) or until theappropriate conditions exist (for example, until

after fertilization).



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Post-translational modification.

Eukaryotic polypeptides must oftenbe processed to yield functional

proteins. Regulation may occur at any of these

steps.For example, cystic fibrosis results from

mutations in the genes for a chloride ionchannel protein that prevents it fromreaching the plasma membrane.

The defective protein is rapidly

degraded.



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The cell limits the lifetimes of normalproteins by selective degradation. Many proteins, like the cyclins in the cell cycle,

must be short-lived to function appropriately.

Proteins intended for degradation aremarked by the attachment ofubiquitinproteins recognized by proteasomes.



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Multi-level Gene Regulation

Transcription Primary level ofregulation

Global (chromosomal, chromatin, loops)Local (genic: sequence elements,

methylation, structural proteins)

Transcript processing and modification

RNA transport

Transcript stability


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Gene Expression Tools

Protein PAGE, Western blotting

RNA Northern blots, RNAfootprinting

DNA recombinant DNA, sequencing& footprinting; Southern blots;restriction & other mapping

Recombinant librariesIn vitro transcription & splicing

Transfection: stable and transient

Transgenic animals/knockouts

9 - Gene Expression 3 - Regulation

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