CHAPTER 12 The Cell Nucleus and the Control of Gene Expression 1.

CHAPTER 12

The Cell Nucleus and the Control of Gene Expression

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• Cells express their genetic information selectively.

• Gene expression is controlled by regulatory machinery in the cell nucleus.

Gene regulation

-Prokaryotes

-Eukaryotes---Genomic control---Transcriptional control---Posttranscriptional control

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12.2 Control of Gene Expression in Bacteria

Genes--Constitutive. (i.e., organic carbon, nitrogen, phosphorus, sulfur and metal ions-iron-,

Ribosomal and glycolysis genes).

---Regulated. (i.e., 02 -metabolic genes-)---Adaptative.(i.e., present or absence of

nutrients)

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Bacterial cells selectively express genes to use the available resources effectively.

The presence of lactose in the medium indices the synthesis of the enzyme β-galactosidase.

The presence of tryptophan in the medium represses the genes that encode enzymes for tryptophan synthesis.

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The kinetics of β-galactosidase induction

Inducible enzymes

Substrate induction

Catabolic Pathway5

Product inhibition

End-product repression

Negative feedback

Anabolic Pathway

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Control of Gene Expression in Bacteria• The Bacterial Operon

– An operon is a functional complex of genes containing the information for enzymes of a metabolic pathway. It includes:

• Structural genes – code for the enzymes and are translated from a single mRNA (Polycistronic).

• Promoter – where the RNA polymerase binds.• Operator – site next to the promoter , where the regulatory

protein can bind.• A repressor (~proteins) which binds to a specific DNA sequence

to determine whether or not a particular gene is transcribed.• The regulatory gene encodes the repressor protein

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Organization of a bacterial operon

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Gene regulation by operons

Inducible operon

Allosteric protein

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Control of Gene Expression in Bacteria (4)

• The lac Operon– It is an inducible operon, which is turned on in the

presence of lactose (inducer).• The lac operon contains three structural genes.• Lactose binds to the repressor, changing its

conformation and making it unable to bind to the operator.

• A repressor protein can bind to the operator and prevent transcription in the absence of lactose.

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The cAMP Receptor Protein (CRP) and Its Function

[Glucose] [cAMP]

Glucose indirectly inhibits the adenyl cyclase

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Repressible operon

The Tryptophan (trp) Operon of E. coli

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• The trp Operon– It is a repressible operon, which is turned off in the

presence of tryptophan.– The trp operon repressor is active only when it is

bound to a corepressor such as tryptophan.

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• Riboswitches– A number of bacterial mRNAs can bind to a small

metabolite (molecules) , which in turn alters the gene involved in the production of such metabolite.

– These mRNAs are called riboswitches because they undergo a conformational change and can suppress gene expression.

– Riboswitches allow bacteria to regulate gene expression in response to some metabolites.

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Riboswitches= small molecules regulating gene expression by interacting mRNA

Prokaryotes and plants

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Control of Gene Expression in Eukaryotes (2)

• Genes are turned on and off as a result of interaction with regulatory proteins.– Each cell type contains a unique

set of proteins.– Regulation of gene expression

occurs on three levels:• Transcriptional-level control• Processing-level control• Translational-level control

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-Eukaryotes---Genomic control---Transcriptional control

•Transcriptional-level control•Processing-level control

---Posttranscriptional control

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-Eukaryotes---Genomic control

----- gene amplification(i.e., in same type of cancers--EGFR, IGFR)-----gene deletion(i.e., red cells no DNA)-----gene rearrangements(i.e., IgG)-----Chromatin (de)condensation-----DNA metylation-----Histone acetylation

---Transcriptional control---Posttranscriptional control

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The cell nucleus

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12.1 The Nucleus of a Eukaryotic Cell (1)• The contents of the nucleus are enclosed by

the nuclear envelope.• A typical non-dividing nucleus includes:

– Chromosomes as extended fibers of chromatin.– Nucleoli for rRNA synthesis.– Nucleoplasm as the fluid where solutes are

dissolved.– The nuclear matrix, which is the protein-

containing fibrillar network.

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The Nucleus of a Eukaryotic Cell (2)

• The Nuclear Envelope– The nuclear envelope is a structure that divides

the nucleus from its cytoplasm.• It consists of two membranes separated by a nuclear

space.• The two membranes are fuses at sites forming a

nuclear pore.• The inner surface of the nuclear envelope is lined by

the nuclear lamina.

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The nuclear envelope

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• The nuclear lamina– Support the nuclear envelope.– It is composed of lamins.– The integrity of the nuclear lamina is regulated by

phosphorylation and dephosphorylation.

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The nuclear lamina

Hutchinson–Gilford progeria syndrome: One point mutant in Lamin A/C, abnormal growth and aging

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The Nucleus of a Eukaryotic Cell (4)• The Structure of the Nuclear Pore Complex

and its Role in Nucleocytoplasmic Exchange– Proteins and RNA are transported in and out of

the nucleus.– Nuclear pores contain the nuclear pore complex

(NPC) that appears to fill the pore like a stopper.– NPC is composed of ~30 proteins called

nucleoporins.

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Movement of materials though the nuclear pore

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A model of the vertebrate NPC

Amino acids

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• Proteins synthesized in the cytoplasm are targeted for the nucleus by the nuclear localization signal (NLS).– Proteins with an NLS stretch bind to an NLS

receptor (importin).– Conformation of the NPC changes as the protein

passes through.– RNAs move through the NPCs as RNPs and carry

NES (nuclear export signals) to pass through.

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Importing proteins from the cytoplasminto the nucleus

NLS

NES

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• Chromosomes and Chromatin– Packaging the Genome

• Chromosomes consist of chromatin fibers, composed of DNA and associated proteins.

• Each chromosome contains a single, continuous linear DNA molecule.

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The Nucleus of a Eukaryotic Cell (8)• DNA and histones are organized into repeating subunits called

nucleosomes.

• Each nucleosome includes a core particle of supercoiled DNA containing H2A, H2B, H3, and H4.

• Histone H1 functions as a linker.

• The histone core complex consists of two molecules each of H2A, H2B, H3, and H4 forming an octamer.– The protein component of chromosomes include histones, a group of

highly conserved proteins.– Histones have a high content of basic amino acids.

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Nucleosomal organization of chromatin

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Levels of organization of chromatin

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• Heterochromatin and Euchromatin– Euchromatin: active DNA– Heterochromatin: inactive DNA

• Constitutive heterochromatin remains condensed all the time (centromeres and telomeres). Consists of highly repeated sequences and few active genes (DNA stability).

• Facultative heterochromatin is inactivated during certain phases of the organism’s life. X chromosomes inactivation during mammalian embryogenesis.

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http://en.wikipedia.org/wiki/Embryogenesis

Sensitivity of Active Genes in Chromatin toDigestion with DNase I

-----Chromatin decondensation

Decondensation ~ gene transcription

Hypersensitive sites (DNAase)35


• Histone and DNA modification is one mechanism to alter the character of nucleosomes.

• DNA and histones are held together by noncovalent bonds.

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– The histone code hypothesis states that the activity of a chromatin region depends on the degree of chemical modification of histone tails.

– Histone tail modifications influence chromatin in two ways:

• Serve as docking sites to recruit nonhistone proteins.• Alter the way in which histones of neighboring

nucleosomes interact with one another.37

-----DNA metylation

Methyl groups can be added to the DNA

Specificity: Cytosines5’-CG-3’

Enzyme: methyl transferaseFunction: gene inactivation

Epigenetic changes (i.e., function)----Epigenomic

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-----Histone acetylation

Acetyl groups can be added to the Histones

Specificity: H3 and H4Enzyme: Acetyl transferaseFunction: gene activation

The prefect combination for gene inactivationDNA methylation

H3/H4 de-acetylation and metylation(heterochromatin)

Heterochromatin protein (HP)139

HDAC: histone De-acetylase complex

HAT: histone acetyltrasnferase

http://www.youtube.com/watch?v=4b-NSWm24BA40

The Nucleus of a Eukaryotic Cell (16)• Telomeres

– The end of each chromosome is called a telomere and is distinguished by a set of repeated sequences.

– New repeats are added by a telomerase, a reverse transcriptase that synthesizes DNA from a RNA template.

– Telomeres are required for the complete replication of the chromosome because they protect the ends from being degraded.

– Telomerase activity is thought to have major effects on cell life

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Telomeres

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The role of telomerase

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• The Nuclear Matrix– The nuclear matrix is a

network of protein-containing fibrils.

– It serves as more than a skeleton to maintain the shape of the nucleus and anchoring the machinery involved in nuclear activities.

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Control of Gene Expression in Eukaryotes (2)

• Genes are turned on and off as a result of interaction with regulatory proteins.– Each cell type contains a unique set of proteins.– Regulation of gene expression occurs on three

levels:-Eukaryotes---Genomic control---Transcriptional control

•Transcriptional-level control•Processing-level control

---Posttranscriptional control45

---Transcriptional control-----differential gene

transcription-----control elements

(transcriptional factors)

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12.4 Transcriptional-level control (1)

1. Differential transcription is the most important mechanism by which eukaryotic cells determine which proteins are synthesized.

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Using a DNA Microarray to Profile the Patterns of Gene Expression in Two Different Cell Types

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Transcriptional-level control (3)

• The Role of Transcription Factors in Regulating Gene Expression– Transcription factors are the proteins that either

acts as transcription activators or transcription inhibitors.

• A single gene can be controlled by different regulatory proteins.

• A single DNA-binding protein may control the expression of many different genes.

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• The Structure of Transcription Factors– Transcription factors

contain a DNA-binding domain and an activation domain.

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1 2

3 4

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Anatomy of a Typical Eukaryotic Gene, with Its Core Promoter and Proximal Control Region

-----control elements

Enhancer

Silencermonocistronic

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Model for Enhancer Action

DHAC: De-acetylase complex MTC: Methyl transferase complex

Co-repressor

HAT: histone acetyl transferaseCo-activator

Remodeling complex

Suppressor or silencer

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Combinatorial Model for Gene Expression

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-Eukaryotes---Genomic control---Transcriptional control

---Post-transcriptional control----alternative splicing----translational----degradation of mRNA----protein degradation

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Figure 26.2

Eukaryotic genes often contain intervening sequencings (introns) that separate the exons and must be removed for proper protein translation to occur.

Removal of introns involves a complicated “molecular device” called a spliceosome that involves protein and RNA (like a ribosome).

Splicing allows an increase in the information content of a genome due to alternative splicing products that produce novel proteins.

Typical mammalian genehas 7-8 exons over ~16 kb

Exons are short (~100-200 bp)Introns are long (~>1kb)

Pre-mRNAmRNASplicing occurs in the nucleus

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Nuclear Splice Junctions Are Short Sequences

• Splice sites are the sequences immediately surrounding the exon–intron boundaries.

• They are named for their positions relative to the intron.

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• The 5 splice site at the 5 (left) end of the intron includes the consensus sequence ′ ′GU.

• The 3 splice site at the 3 (right) end of the intron includes the consensus ′ ′sequence AG.

• The GU-AG rule describes the requirement for these constant dinucleotides at the first two and last two positions of introns in pre-mRNAs.

Figure 26.3

Splice sites are generic.

GT…AG (DNA)-(~98 %);(~2% ?)

Directional (one direction)

The apparatus for splicing is not tissue specific (with some exceptions).

The mechanism by which pairs of splice sites are correctly identified is unknown.

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12.7 Post-translational Control: Determining Protein Stability (1)

• The factors that control a protein’s lifetime are not well understood.

• Protein stability may be determined by the amino acids on the N-terminus.

• Degradation of proteins is carried out within hollow, cylindrical proteasomes.

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Post-translational Control: Determining Protein Stability (2)

• Proteasomes recognize proteins linked to ubiquitin ( small protein)

• Ubquitin is transferred by ubiquitin ligases to proteins being degraded.

• Once polubiquitanated, a protein is recognized by the cap of the proteasome.

• Once degraded, the component amino acids are released back into the cytosol.

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Proteasome structure

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CHAPTER 12 The Cell Nucleus and the Control of Gene Expression 1.

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