16 Regulation of Gene Expression. 16 Regulation of Gene Expression 16.1 How Is Gene Expression...

16Regulation of Gene

Expression

16 Regulation of Gene Expression

16.1 How Is Gene Expression Regulated in Prokaryotes?

16.2 How Is Eukaryotic Gene Transcription Regulated?

16.3 How Do Viruses Regulate Their Gene Expression?

16.4 How Do Epigenetic Changes Regulate Gene Expression?

16.5 How Is Eukaryotic Gene Expression Regulated After Transcription?

16 Regulation of Gene Expression

Behavioral epigenetics: study of heritable changes in gene expression that do not involve changes in the DNA sequence.

Methylation of some gene promoters may result from high levels of stress, and inhibit gene transcription. Methylation in the glucocorticoid receptor gene may result in behavioral problems.

Opening Question:

Can epigenetic changes be manipulated?

Prokaryotes can make some proteins only when they are needed. To shut off supply of a protein, the cell can:

• Downregulate mRNA transcription

• Hydrolyze mRNA, preventing translation

• Prevent mRNA translation at the ribosome

• Hydrolyze the protein after it is made

• Inhibit the protein’s function

The earlier the cell can stop protein synthesis, the less energy is wasted.

Blocking transcription is more efficient than transcribing the gene, translating the message, and then degrading or inhibiting the protein.

Gene expression begins at the promoter.

Two types of regulatory proteins can bind to promoters:

• Negative regulation—a repressor protein prevents transcription

• Positive regulation—an activator protein stimulates transcription

Figure 16.1 Positive and Negative Regulation

E. coli in the human intestine must adjust quickly to changes in food supply.

Glucose is the easiest sugar to metabolize.

Lactose is β-galactoside (disaccharide of galactose and glucose).

Three proteins are need for the uptake and metabolism of lactose.

-galactoside permease—carrier protein that moves lactose into the cell

-galactosidase—hydrolyses lactose

-galactoside transacetylase—transfers acetyl groups from acetyl CoA to certain -galactosides

If E.coli is grown with glucose but no lactose, no enzymes for lactose conversion are produced.

If lactose is predominant and glucose is low, E.coli synthesizes all three enzymes after a short lag period.

Figure 16.2 An Inducer Stimulates the Expression of a Gene for an Enzyme

During the lag period, mRNA for β-galactosidase is produced.

If lactose is removed, the mRNA level goes down.

Compounds that stimulate protein synthesis are called inducers;

The proteins are inducible proteins.

Constitutive proteins are made at a constant rate.

Metabolic pathways can be regulated in two ways:

• Allosteric regulation of enzyme-catalyzed reactions allows rapid fine-tuning

• Regulation of protein synthesis is slower but conserves energy and resources. Protein synthesis requires a lot of energy

Figure 16.3 Two Ways to Regulate a Metabolic Pathway

Structural genes specify primary protein structure—the amino acid sequence.

The 3 structural genes for lactose enzymes are adjacent on the chromosome and share a promoter, forming the lac operon.

An operon is a gene cluster with a single promoter.

A typical operon consists of:

• A promoter

• Two or more structural genes

• An operator—a short sequence between the promoter and the structural genes; binds to regulatory proteins

Figure 16.4 The lac Operon of E. coli

Three ways to control operon transcription:

• An inducible operon regulated by a repressor protein

• A repressible operon regulated by a repressor protein

• An operon regulated by an activator protein

In the lac operon the operator can bind a repressor protein, which blocks transcription.

The repressor has 2 binding sites: one for the operator, and one for the inducer (lactose).

When lactose is absent, the repressor prevents binding of RNA polymerase to the promoter.

Figure 16.5 The lac Operon: An Inducible System (Part 1)

When lactose is present, it binds to the repressor and changes the repressor’s shape.

This prevents the repressor from binding to the operator, and then RNA polymerase can bind to the promoter, and the genes are transcribed.

Figure 16.5 The lac Operon: An Inducible System (Part 2)

Other E. coli systems are repressible—the operon is turned on unless repressed under specific conditions.

In these systems, the repressor isn’t bound to the operator until a co-repressor binds to it.

The repressor then changes shape, binds to the operator, and blocks transcription.

The trp operon is a repressible system.

The genes code for enzymes that catalyze synthesis of tryptophan.

When there is enough tryptophan in the cell, tryptophan binds to the repressor, which then binds to the operator. Tryptophan is the co-repressor.

Inducible systems: metabolic substrate (inducer) interacts with a regulatory protein (repressor); repressor can’t bind to operator and transcription proceeds.

Repressible systems: a metabolic product (co-repressor) binds to a regulatory protein, which then binds to the operator and blocks transcription.

Inducible systems control catabolic pathways—they are turned on when substrate is available.

Repressible systems control anabolic pathways—they are turned on until product concentration becomes excessive.

Positive control: an activator protein can increase transcription.

If glucose and lactose levels are both high, the lac operon is not transcribed efficiently.

Efficient transcription requires binding of an activator protein to its promoter.

If glucose levels are low, a signaling pathway leads to increased levels of cyclic AMP.

cAMP binds to cAMP receptor protein (CRP); conformational change in CRP allows it to bind to the lac promoter.

CRP is an activator of transcription; its binding results in more efficient binding of RNA polymerase and thus increased transcription.

Figure 16.6 Catabolite Repression Regulates the lac Operon

If glucose is abundant, CRP does not bind to the lac operon promoter and efficiency of transcription is reduced.

This is catabolite repression, a system of gene regulation in which presence of a preferred energy source represses other catabolic pathways.

Figure 16.6 Catabolite Repression Regulates the lac Operon

Table 16.1

Promoters bind and orient RNA polymerase so that the correct DNA strand is transcribed.

All promoters have consensus sequences that allow them to be recognized by RNA polymerase.

Different classes of consensus sequences are recognized by regulatory proteins called sigma factors.

Sigma factors bind to RNA polymerase and direct it to certain promoters.

Genes for proteins with related functions may be at different locations in the genome, but share consensus sequences and can be recognized by sigma factors.

Sigma-70 factor is active most of the time and binds to consensus sequences of housekeeping genes (genes normally expressed in actively growing cells).

Others are activated only under specific conditions.

In development of multicellular organisms, certain proteins must be made at just the right times and in just the right cells.

The expression of eukaryotic genes must be precisely regulated.

Regulation can occur at several different points.

Figure 16.7 Potential Points for the Regulation of Gene Expression (Part 1)

Figure 16.7 Potential Points for the Regulation of Gene Expression (Part 2)

Both prokaryotes and eukaryotes use DNA-protein interactions and negative and positive control to regulate gene expression.

But there are differences, some dictated by the presence of a nucleus, which physically separates transcription and translation.

Table 16.2

Eukaryote promoters contain a sequence called the TATA box—where DNA begins to denature.

Promoters also include regulatory sequences recognized by transcription factors (regulatory proteins).

RNA polymerase II can only bind to the promoter after general transcription factors have assembled on the chromosome:

TFIID binds to TATA box; then other factors bind to form an initiation complex.

Figure 16.8 The Initiation of Transcription in Eukaryotes (Part 1)

Figure 16.8 The Initiation of Transcription in Eukaryotes (Part 2)

Some regulatory sequences are common to promoters of many genes, such as the TATA box.

Some sequences are specific to a few genes and are recognized by transcription factors found only in certain tissues.

These play an important role in cell differentiation.

Enhancers: regulatory sequences that bind transcription factors that activate transcription or increase rate of transcription.

Silencers: bind transcription factors that repress transcription.

Most regulatory sequences are located near the transcription start site.

Others may be located thousands of base pairs away. Transcription factors may interact with the RNA polymerase complex and cause the DNA to bend.

Figure 16.9 Transcription Factors and Transcription Initiation

Often there are many transcription factors involved.

The combination of factors present determines the rate of transcription.

Although the same genes are present in all cells, the fate of the cell is determined by which of its genes are expressed.

Transcription factors have common structural motifs in the domains that bind to DNA.

A common motif is helix-turn-helix:

For DNA recognition, the structural motif must:

• Fit into a major or minor groove

• Have amino acids that can project into interior of double helix

• Have amino acids that can bond with interior bases

Many repressor proteins, such as the lac repressor, have helix-turn-helix motifs:

During development, cell differentiation is often mediated by changes in gene expression.

All differentiated cells contain the entire genome; their specific characteristics arise from differential gene expression.

Cellular therapy is a new approach to diseases that involve degeneration of one cell type.

Alzheimer’s disease involves degeneration of neurons in the brain.

If other cells could be made to differentiate into neurons, they could be transferred to the patient.

Figure 16.10 Expression of Specific Transcription Factors Turns Fibroblasts into Neurons

How do eukaryotes coordinate expression of sets of genes?

Most have their own promoters, and may be far apart in the genome.

If the genes have common regulatory sequences, they can be regulated by the same transcription factors.

Plants in drought stress must synthesize several proteins (the stress response). The genes are scattered throughout the genome.

Each of the genes has a regulatory sequence called stress response element (SRE). A transcription factor binds to this element and stimulates mRNA synthesis.

Figure 16.11 Coordinating Gene Expression

Working with Data 16.1: Expression of Transcription Factors Turns Fibroblasts into Neurons

To determine whether specific transcription factors might change one type of cell to another, genes for transcription factors in neurons were inserted into fibroblasts.

When five transcription factors were introduced into fibroblasts and expressed from very strong promoters, the fibroblasts became neurons.

Three main criteria were used to determine that the transformed cells were neurons:

• Morphology

• Electrical excitability

• Lack of cell division

Question 1:

Neurons respond to electrical stimulation by generating an action potential. The electrical activity of a stimulated transformed fibroblast cell is shown in Fig. A: 8, 12, and 20 days after addition of the transcription factors.

What is the magnitude of the action potential of the transformed cell in millivolts?

Look at Figure 45.10. How does this compare?

Working with Data 16.1, Figure A

Figure 45.10 The Course of an Action Potential

Question 2:

The rate of cell division in the population of transformed cells was measured by the incorporation of the labeled nucleotide BrdU into their DNA.

The percentage of labeled—and hence dividing—cells is shown in Fig. B.

Did cell division stop in the transformed cells? Explain your answer.

Working with Data 16.1, Figure B

Viruses are infectious agents that infect cellular organisms, and can’t reproduce outside their host cells.

A bacterial virus (bacteriophage) injects its genetic material into a host cell and turns that cell into a virus factory.

Other viruses enter cells and then shed their coats and take over the cell’s replication machinery.

Virus particles, called virions, consist of DNA or RNA, a protein coat, and sometimes a lipid envelope.

Viral genomes contain sequences that encode regulatory proteins that “hijack” the host cells’ transcriptional machinery.

The viral lytic cycle—host cell lyses and releases progeny viruses.

A phage injects a host cell with genetic material that takes over synthesis.

New phage particles appear rapidly and are soon released from the lysed cell.

Figure 16.12 Bacteriophage and Host

The lytic cycle has two stages.

1. Early stage: viral promoter binds host RNA polymerase. Viral genes adjacent to this promoter are transcribed (positive regulation).

Early genes encode proteins that shut down host transcription (negative regulation) and stimulate viral genome replication and transcription of viral late genes (positive regulation).

Three minutes after DNA entry, viral nuclease enzymes digest the host’s chromosome, providing nucleotides for the synthesis of viral genomes.

Figure 16.13 The Lytic Cycle: A Strategy for Viral Reproduction

2. Late stage: viral late genes are transcribed (positive regulation).

They encode the viral capsid proteins and enzymes to lyse the host cell.

The whole process from binding and infection to release of new particles takes about 30 minutes.

Some viruses have evolved lysogeny—the lytic cycle is delayed.

Viral DNA integrates with the host DNA to form a prophage.

As the host cell divides, the viral DNA replicates too and can last for thousands of generations.

Figure 16.14 The Lytic and Lysogenic Cycles of Bacteriophages

If a host cell is not growing well, the virus may switch to the lytic cycle.

The prophage excises itself from the host chromosome and reproduces.

Understanding the regulation of gene expression that underlies the lysis/lysogeny switch was a major achievement.

How does the prophage “know” when to switch?

Two virus genes encode regulatory proteins cI and Cro.

cI blocks expression of genes for the lytic cycle and promotes expression of genes for lysogeny; Cro has the opposite effect.

If conditions are favorable for host cell growth, cI accumulates and outcompetes Cro for DNA binding; phage enters lysogenic cycle.

If host cell is under stress, cI is degraded and no longer blocks expression of Cro; phage enters lytic cycle.

Figure 16.15 Control of Bacteriophage Lysis and Lysogeny

cI protein is degraded because it is structurally similar to E. coli protein LexA that is also degraded.

LexA represses DNA repair mechanisms under normal conditions, but is degraded by other proteins when the cell is stressed.

Eukaryote viruses:

DNA viruses: Double- or single-stranded (complementary strand is made in the host cell)

Some have both lytic and lysogenic life cycles.

Examples: Herpes viruses and papillomaviruses (warts).

RNA viruses: Usually single-stranded

RNA is translated by the host cell to make viral proteins involved in RNA replication.

Example: Influenza virus.

Retroviruses: RNA virus with a gene for reverse transcriptase—synthesizes DNA from an RNA template.

The DNA copy is inserted into the host genome.

Example: Human immunodeficiency virus (HIV).

HIV regulation occurs at the elongation stage of transcription.

HIV is an enveloped virus—enclosed in a phospholipid membrane derived from the host.

The envelope fuses with the host cell membrane, the virus enters, and its capsid is broken down.

Figure 16.16 The Reproductive Cycle of HIV

Reverse transcriptase uses the viral RNA to make a complementary DNA (cDNA) strand.

A copy of the cDNA is also made, and the double-stranded cDNA is inserted into host chromosome by integrase. The inserted DNA is called a provirus.

The provirus resides permanently in the host chromosome, and can be inactive (latent) for years.

Transcription of viral DNA is initiated, but host cell proteins prevent elongation.

Figure 16.17 Regulation of Transcription by HIV (Part 1)

Under certain conditions, transcription initiation increases, and some viral RNA is made, including RNA for a protein called tat (transactivator of transcription).

tat binds to the viral RNA and production of full-length viral RNA is dramatically increased. The rest of the viral life cycle then proceeds.

Figure 16.17 Regulation of Transcription by HIV (Part 2)

Nearly every step in the HIV life cycle is a potential target for anti-HIV drugs:

• Reverse transcriptase inhibitors (step 2)

• Integrase inhibitors (step 3)

• Protease inhibitors block posttranslational processing of viral proteins (step 5)

Combinations of drugs have been very successful at treating HIV infection, but new strains rapidly emerge.

New drugs are being developed to target other life cycle steps, including drugs that interfere with binding of virus to host cell, and interfere with tat activity.

Epigenetics is the study of changes in gene expression that occur without changes in the DNA sequence.

These changes are reversible, but sometimes stable and heritable.

Includes two processes: DNA methylation and chromosomal protein alterations.

Methylation:

• A methyl group is covalently added to the 5′ carbon of cytosine, forming 5-methylcytosine

• Catalyzed by DNA methyltransferase

• Usually occurs in regions rich in C and G doublets, called CpG islands—often in promoters

It can be heritable: when DNA replicates, a maintenance methylase catalyzes formation of 5-methylcytosine in the new strand.

Or, the methylation pattern may be altered because it is reversible.

Demethylase catalyzes removal of methyl groups.

Figure 16.18 DNA Methylation: An Epigenetic Change (Part 1)

Figure 16.18 DNA Methylation: An Epigenetic Change (Part 2)

Effects of DNA methylation:

• Methyl groups in promoter regions attract proteins for transcription repression. Methylated genes are often inactive

• In development, early demethylation allows many genes to become active

Later, some genes may be “silenced” by methylation

Silent genes may be turned back on:

DNA methylation can play a role in cancer—oncogenes get activated and promote cell division, and tumor suppressor genes can be turned off.

Chromosomal protein alterations or chromatin remodeling:

DNA is packaged with histone proteins into nucleosomes. The DNA is inaccessible to RNA polymerase and transcription factors.

The histones have “tails” with positively charged amino acids, which are attracted to negatively charged DNA.

Histone acetyltransferases add acetyl groups to the tails which changes their charges, and opens up the nucleosome to activate transcription.

Figure 16.19 Epigenetic Remodeling of Chromatin for Transcription

Histone deacetylases removes the acetyl groups, which represses transcription.

In some cancers, genes that inhibit cell division are excessively deacetylated.

Drugs that inhibit histone deacetylase may be useful to treat the cancer.

Histones can also be modified by:

• Methylation—inactivates genes

• Phosphorylation—effects depend on which amino acids are involved

All the epigenetic effects are reversible, so gene activity may be determined by very complex patterns of histone modification.

Environmental factors can induce epigenetic changes:

Monozygotic (identical) twins have identical genomes, and have been used to study epigenetic effects.

In 3-year-old twins, DNA methylation patterns are the same. By age 50, when twins have been living apart in different environments, methylation patterns were quite different.

Genomic imprinting:

In mammals, eggs and sperm develop different methylation patterns.

For about 200 genes, offspring inherit an inactive (methylated) copy and an active (demethylated) one.

Figure 16.20 Genomic Imprinting

Example of imprinting: a region on human chromosome 15 called 15q11

Rarely, a chromosome deletion results in the baby having only the male or female version of the gene.

• Male pattern results in Angelman syndrome, with epilepsy, tremors, and constant smiling

• Female pattern results in Prader-Willi syndrome, marked by muscle weakness and obesity

The gene sequences are the same in both cases; the epigenetic patterns are different.

Patterns of DNA methylation may include large regions or whole chromosomes.

Two kinds of chromatin:

• Euchromatin—diffuse, light-staining; contains DNA that is transcribed

• Heterochromatin—condensed, dark-staining, contains genes not transcribed

One type of heterochromatin is the inactive X chromosome in mammals.

Males (XY) and females (XX) contain different numbers of X-linked genes, yet for most genes transcription rates are similar.

Early in development, one of the X chromosomes in females is inactivated.

Which X chromosome gets inactivated is random in each cell.

The inactivated X chromosome is heterochromatin, and shows up as a Barr body in human female cells.

The DNA is heavily methylated, and unavailable for transcription, except for the Xist gene.

Figure 16.21 X Chromosome Inactivation

RNA transcribed from Xist (X inactivation-specific transcript) binds to the chromosome, spreading the inactivation.

This RNA is an example of interference RNA.

Figure 16.21 X Chromosome Inactivation

After transcription, eukaryotic gene expression can be regulated in the nucleus before mRNA export, or after mRNA leaves.

Control mechanisms include alternative splicing of pre-mRNA, gene silencing, translation repressors, and regulation of protein breakdown.

Alternative splicing: different mRNAs can be made from the same gene.

Introns are spliced out; mature mRNAs have none.

Sometimes exons are spliced out too—resulting in different proteins.

There are many more human mRNAs than there are coding genes.

Figure 16.22 Alternative Splicing Results in Different Mature mRNAs and Proteins

MicroRNAs(miRNAs): small RNAs produced by noncoding regions of DNA.

First found in C. elegans. Two genes effect transition through the larval stages:

• Mutations in lin-14 caused the worm to skip the 1st stage; normal role is to facilitate stage 1 events.

• lin-4 mutations caused some cells to repeat a development pattern normally shown in the 1st stage; its normal role is negative regulation of lin-14.

lin-14 encodes a transcription factor that affects genes involved in larval cell progression.

lin-4 encodes a 22-base miRNA that inhibits lin-14 expression post-transcriptionally, by binding to its mRNA.

The human genome has about 1,000 miRNA encoding regions.

miRNAs can inhibit translation by binding to target mRNAs. Each one is about 22 bases long and has many targets, as binding doesn’t have to be perfect.

Figure 16.23 mRNA Inhibition by RNAs (Part 1)

Small interfering RNAs (siRNAs) also result in RNA silencing.

Often arise from viral infection and transposon sequences. They bind to target mRNA and cause its degradation.

May have evolved as defense to prevent translation of viral and transposon sequences.

Figure 16.23 mRNA Inhibition by RNAs (Part 2)

Cells have two major ways to control the amount of protein after transcription:

• Block mRNA translation

• Alter how long new proteins persist in the cell

Translation can be altered by:

• miRNAs that inhibit translation

• GTP cap on 5′ end of mRNA can be modified—if cap is unmodified mRNA is not translated

• Repressor proteins can block translation directly

Translational repressor proteins can bind to noncoding regions of mRNA and block translation by preventing it from binding to a ribosome.

The RNA region that is bound by the repressor is called a riboswitch.

Figure 16.24 Translational Repressor Can Repress Translation

Protein longevity:

Protein content of a cell is a function of synthesis and degradation.

Proteins can be targeted for destruction when ubiquitin attaches to it and attracts other ubiquitins, forming a polyubiquitin chain.

The complex binds to a proteasome—a large complex where the ubiquitin is removed and the protein is digested by proteases.

Figure 16.25 A Proteasome Breaks Down Proteins

Some strains of human papillomavirus (HPV) add ubiquitin to p53 and retinoblastoma proteins, targeting them for degradation.

These proteins normally inhibit the cell cycle, so the result of this HPV activity is unregulated cell division (cancer).

16 Answer to Opening Question

Epigenetic changes often involve methylation.

Some nutrients, such as folic acid, have methyl groups and participate in DNA modification.

Experiments with mice show that diets rich in these nutrients change epigenetic patterns that remain throughout life.

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