Eukaryotic Gene ExpressionEukaryotic Gene Expression

Managing the Complexities of Managing the Complexities of Controlling Eukaryotic GenesControlling Eukaryotic Genes

Prokaryotes vs. EukaryotesProkaryotes vs. EukaryotesProkaryotesProkaryotes EukaryotesEukaryotes

Closely related genes are Closely related genes are clustered togetherclustered together

Related genes are located on Related genes are located on different chromosomesdifferent chromosomes

Largely transcriptional Largely transcriptional controlcontrol

Significant transcriptional Significant transcriptional control; other levels of control control; other levels of control possiblepossible

Larger number of larger-sized Larger number of larger-sized genesgenes

Trans-acting sequence-Trans-acting sequence-specific DNA binding specific DNA binding proteinsproteins

Trans-acting sequence-Trans-acting sequence-specific DNA binding proteinsspecific DNA binding proteins

Proximal Cis-acting Proximal Cis-acting sequencessequences

Cis-acting sequences can be Cis-acting sequences can be located at significant located at significant distancesdistances

Control Points for Control Points for Gene Expression in EukaryotesGene Expression in Eukaryotes

DNADNA

RNARNA

ProteinProtein

transcriptiontranscription

translationtranslation

Transcriptional ControlTranscriptional Control

Translational ControlTranslational Control

Post-Translational ControlPost-Translational Control

Post-Transcriptional ControlPost-Transcriptional Control

Levels of Eukaryotic Chromatin StructureLevels of Eukaryotic Chromatin Structure

First level of chromatin coiling Nucleosome = DNA + histone proteins

Variations in chromatin condensation affect gene activity.Variations in chromatin condensation affect gene activity.

Transcriptional Regulation: Transcriptional Regulation: Effects of Chromatin StructureEffects of Chromatin Structure

Decompaction of chromatin: Decompaction of chromatin: • Transcription factors unwindTranscription factors unwind

nucleosomes in the area where nucleosomes in the area where transcription will begin, creating transcription will begin, creating DNAse I hypersensitive sites DNAse I hypersensitive sites

• RNA polymerase unwinds moreRNA polymerase unwinds more nucleosomes as transcription nucleosomes as transcription proceeds proceeds

Transcriptional Regulation: Transcriptional Regulation:

Effects of Chromatin StructureEffects of Chromatin Structure Acetylation of histone proteins (adding -CHAcetylation of histone proteins (adding -CH33CO) CO)

reduces DNA-histone interaction, permitting reduces DNA-histone interaction, permitting transcription factors to bind. transcription factors to bind.

Transcriptional Regulation: Transcriptional Regulation: Effects of Chromatin StructureEffects of Chromatin Structure

DNA MethylationDNA Methylation• DNA Methylation (adding -CHDNA Methylation (adding -CH33) can occur on ) can occur on

cytosines at CpG groupings near transcription cytosines at CpG groupings near transcription start sites start sites

•Inactive genes have methylated cytosines Inactive genes have methylated cytosines •Active genes have demethylated cytosinesActive genes have demethylated cytosines

• Acetylation of histones is associated withAcetylation of histones is associated with cytosine demethylation cytosine demethylation

Transcriptional Regulation: Control of InitiationTranscriptional Regulation: Control of Initiation

•Transcriptional Activator Proteins assist in the Transcriptional Activator Proteins assist in the formation or action of the basal transcription formation or action of the basal transcription apparatus apparatus

Transcriptional Regulation: Control of InitiationTranscriptional Regulation: Control of Initiation

• Transcriptional Activator Proteins bind to Transcriptional Activator Proteins bind to Enhancer sequences that increase Enhancer sequences that increase transcriptiontranscription– Enhancers can influence Enhancers can influence

promoters at distances promoters at distances of 50 kb or greater of 50 kb or greater due to DNA looping due to DNA looping mechanismmechanism

– Insulators control the direction of enhancer actionInsulators control the direction of enhancer action

Transcriptional Regulation: Control of InitiationTranscriptional Regulation: Control of Initiation

• Transcriptional Repressor Proteins have three Transcriptional Repressor Proteins have three possible modes of action possible modes of action

– compete with activators for DNA binding compete with activators for DNA binding sitessites

– bind to sites near activator site andbind to sites near activator site and inhibit inhibit activator contact with basal transcription activator contact with basal transcription apparatusapparatus

– interfere with assembly of basal transcription interfere with assembly of basal transcription apparatusapparatus

Applying Your KnowledgeApplying Your Knowledge

What effect does each of the following have on What effect does each of the following have on transcription? transcription?

• Methylation of Cytosines in CpG groups near Methylation of Cytosines in CpG groups near transcription initiation sitestranscription initiation sites

• Binding of an Activator Protein to an Enhancer Binding of an Activator Protein to an Enhancer SequenceSequence

• Acetylation of Histones in the nucleosomes Acetylation of Histones in the nucleosomes covering a gene sequencecovering a gene sequence

Thumbs Up: Increases TranscriptionThumbs Up: Increases TranscriptionThumbs Down: Decreases TranscriptionThumbs Down: Decreases TranscriptionThumbs Horizontal: No change Thumbs Horizontal: No change

Post-Transcriptional Regulation: Post-Transcriptional Regulation:

Alternative RNA SplicingAlternative RNA Splicing

Post-Transcriptional Regulation: Post-Transcriptional Regulation: RNA EditingRNA Editing

Base substitution after transcription =

Translational Regulation: Translational Regulation: RNA StabilityRNA Stability

• Degradation of mRNA can occur from the Degradation of mRNA can occur from the 5’ or 3’ end5’ or 3’ end

• Stability of mRNA depends on Stability of mRNA depends on – 5’ cap5’ cap– 3’ poly-A tail3’ poly-A tail– 5’ and 3’ UTRs: serve as binding sites for 5’ and 3’ UTRs: serve as binding sites for

regulatory factorsregulatory factors– Coding regionCoding region

• Example: Hormone prolactin increases Example: Hormone prolactin increases the longevity of casein mRNA coding for the longevity of casein mRNA coding for milk protein in lactating mammalsmilk protein in lactating mammals

Translational RegulationTranslational Regulation

• Masking of mRNAsMasking of mRNAs– Many species store mRNAs in the cytoplasm Many species store mRNAs in the cytoplasm

of the egg. These mRNAs are inactive due of the egg. These mRNAs are inactive due to masking by proteins. Fertilization of the to masking by proteins. Fertilization of the egg initiates unmasking and translation of egg initiates unmasking and translation of these mRNAs.these mRNAs.

• Availability of specific tRNAsAvailability of specific tRNAs– In the embryonic development of a In the embryonic development of a

hornworm, an mRNA is present from day 1 hornworm, an mRNA is present from day 1 but a specific tRNA needed for its translation but a specific tRNA needed for its translation is not produced until day 6.is not produced until day 6.

Translational Regulation:Translational Regulation:RNA SilencingRNA Silencing

Small interfering

RNAs

RNA-induced silencing complex

microRNAs

Post-Translational Modification: Post-Translational Modification: PhosphorylationPhosphorylation

Addition or removal of a Addition or removal of a phosphate group is a phosphate group is a common way to change common way to change protein activity. protein activity.

Post-Translational Modification: Post-Translational Modification: Peptide cleavagePeptide cleavage

Proteins that have an inactive form after Proteins that have an inactive form after

synthesis are activated by removal of a synthesis are activated by removal of a small number of amino acids. small number of amino acids. ProthrombinProthrombin

ThrombinThrombin

Cleavage

FibrinFibrinpolymer polymer (blood clot)(blood clot)

FibrinogenFibrinogen

FibrinFibrin

Cleavage

Activation of blood clotting factors by cleavage

Applying Your KnowledgeApplying Your Knowledge

Which type of control is demonstrated by Which type of control is demonstrated by • Alternative RNA splicing mechanisms that give rise Alternative RNA splicing mechanisms that give rise

to different protein products? to different protein products? • Addition of a phosphate group to activate a protein?Addition of a phosphate group to activate a protein?• Increased stability of mRNA in the presence of a Increased stability of mRNA in the presence of a

regulator molecule? regulator molecule? • Binding of a repressor protein to the regulatory Binding of a repressor protein to the regulatory

promoter so that an activator protein is unable to promoter so that an activator protein is unable to bind to this site?bind to this site?

1.1. Transcriptional ControlTranscriptional Control2.2. Post-Transcriptional ControlPost-Transcriptional Control3.3. Translational ControlTranslational Control4.4. Post-Translational Control Post-Translational Control