Regulation of Gene Expression-Chapter 15

• Prokaryotes and eukaryotes alter gene expression in response to their changing environment

• In multicellular eukaryotes, gene expression also regulates development and is responsible for differences in cell types

• Protein/DNA, RNA/DNA, and RNA/RNA interactions all play roles in regulating gene expression in eukaryotes

Bacteria often respond to environmental change by regulating transcription

• Natural selection has favored bacteria that produce only the products needed by that cell

• A cell can regulate the production of enzymes by feedback inhibition or by gene regulation

• Which of these forms of regulation saves more energy? Which can respond to changes more quickly?

LE 18-20

Regulation of enzymeactivity

Regulation of enzymeproduction

Enzyme 1

Regulation of gene expression

Enzyme 2

Enzyme 3

Enzyme 4

Enzyme 5

Gene 2

Gene 1

Gene 3

Gene 4

Gene 5

Tryptophan

Precursor

Feedbackinhibition

The Operon Model

• Gene expression in bacteria is controlled by the operon model

• Jacob and Monod (1961)

• Operon elements-repressor gene; operator; structural gene(s)

• Inducible and repressible operons

Operons: The Basic Concept

• A group of functionally related genes can be coordinately controlled by a single “on-off switch”

• The regulatory “switch” is a segment of DNA called an operator usually positioned within the promoter

• An operon is the entire stretch of DNA that includes the operator, the promoter, and the genes that they control

Repressible and Inducible Operons: Two Types of Negative Gene Regulation

• A repressible operon is one that is usually on; binding of a repressor to the operator shuts off transcription

• The trp operon is a repressible operon

• An inducible operon is one that is usually off; a molecule called an inducer inactivates the repressor and turns on transcription

• The lac operon is an inducible operon

• Inducible enzymes usually function in catabolic pathways; their synthesis is induced by a chemical signal

• Repressible enzymes usually function in anabolic pathways; their synthesis is repressed by high levels of the end product

• Regulation of the trp and lac operons involves negative control of genes because operons are switched off by the active form of the repressor

The repressible operon (trp operon)

• Shuts down a biochemical pathway when the end product of the pathway build up.

• Normal status-transcription occurs because the operator is unblocked.

• End product of the pathway (co-repressor) alters shape of the repressor protein so that it binds to the operator blocking transcription of the structural genes

LE 18-21a

Promoter Promoter

DNA trpR

Regulatorygene

RNApolymerase

mRNA

3

5

Protein Inactiverepressor

Tryptophan absent, repressor inactive, operon on

mRNA 5

trpE trpD trpC trpB trpA

OperatorStart codonStop codon

trp operon

Genes of operon

E

Polypeptides that make upenzymes for tryptophan synthesis

D C B A

LE 18-21b_1

DNA

Protein

Tryptophan(corepressor)

Tryptophan present, repressor active, operon off

mRNA

Activerepressor

LE 18-21b_2

DNA

Protein

Tryptophan(corepressor)

Tryptophan present, repressor active, operon off

mRNA

Activerepressor

No RNA made

• By default the trp operon is on and the genes for tryptophan synthesis are transcribed

• When tryptophan is present, it binds to the trp repressor protein, which then turns the operon off

• The repressor is active only in the presence of its corepressor tryptophan; thus the trp operon is turned off (repressed) if tryptophan levels are high

Inducible operons

• Activates breakdown of a substrate when the substrate becomes available.

• Normal status-transcription does not occurs because the operator is blocked by the repressor protein.

• Substrate (inducer) alters shape of the repressor protein so that does not bind to the operator allowing transcription of the structural genes to occur

• The lac operon is an inducible operon and contains genes that code for enzymes used in the hydrolysis and metabolism of lactose

• By itself, the lac repressor is active and switches the lac operon off

• A molecule called an inducer inactivates the repressor to turn the lac operon on

• For the lac operon, the inducer is allolactose, formed from lactose that enters the cell

LE 18-22a

DNA lacl

Regulatorygene

mRNA

5

3

RNApolymerase

ProteinActiverepressor

NoRNAmade

lacZ

Promoter

Operator

Lactose absent, repressor active, operon off

LE 18-22b

DNA lacl

mRNA5

3

lac operon

Lactose present, repressor inactive, operon on

lacZ lacY lacA

RNApolymerase

mRNA 5

Protein

Allolactose(inducer)

Inactiverepressor

-Galactosidase Permease Transacetylase

Positive Gene Regulation

• The lac operon can be turned on or off by the presence/absence of the inducer molecule (lactose)

• CAP and CAMP can also regulate the rate of transcription of the lac operon (volume control versus on/off control)

• If both glucose and lactose are present, the bacteria will preferentially use glucose

LE 18-23a

DNA

cAMP

lacl

CAP-binding site

Promoter

ActiveCAP

InactiveCAP

RNApolymerasecan bindand transcribe

Operator

lacZ

Inactive lacrepressor

Lactose present, glucose scarce (cAMP level high): abundant lacmRNA synthesized

LE 18-23b

DNA lacl

CAP-binding site

Promoter

RNApolymerasecan’t bind

Operator

lacZ

Inactive lacrepressor

InactiveCAP

Lactose present, glucose present (cAMP level low): little lacmRNA synthesized

Positive Gene Regulation

• E. coli will preferentially use glucose when it is present in the environment

• When glucose is scarce, CAP (catabolite activator protein) acts as an activator of transcription

• CAP is activated by binding with cyclic AMP (cAMP)

• Activated CAP attaches to the promoter of the lac operon and increases the affinity of RNA polymerase, thus accelerating transcription

• When glucose levels increase, CAP detaches from the lac operon, and transcription proceeds at a very low rate, even if lactose is present

• CAP helps regulate other operons that encode enzymes used in catabolic pathways

Differential Gene Expression in Eukaryotes

• Almost all the cells in an multicellular organism are genetically identical

• Differences between cell types result from differential gene expression, the expression of different genes by cells with the same genome

• In a given cell, usually about 20% of its genes are active at a given time (rest turned off)

• Errors in gene expression can lead to diseases including cancer

• Gene expression is regulated at many stages in eukaryotes (in addition to regulation at the transcriptional level which bacteria also do)

Regulation of chromatin Structure:

• DNA is packed into an elaborate complex known as chromatin

• The basic unit of chromtain is the nucleosome• Histone proteins help maintain nucleosome

structure• The structural organization of chromatin packs

DNA into a compact form and also helps regulate gene expression in several ways

Fig. 16-21b

30-nm fiber

Chromatid (700 nm)

Loops Scaffold

300-nm fiber

Replicated chromosome (1,400 nm)

30-nm fiber Looped domains (300-nm fiber)

Metaphase chromosome

Fig. 16-21a

DNA double helix (2 nm in diameter)

Nucleosome(10 nm in diameter)

Histones Histone tailH1

DNA, the double helix Histones Nucleosomes, or “beads on a string” (10-nm fiber)

Chromatin Packing in Eukaryotes

• Loosely packed DNA (euchromatin) is usually transcribed

• Tightly packed DNA (heterochromatin) is not usually transcribed

Regulation of Chromatin Structure

• Chemical modifications to histones and DNA of chromatin influence both chromatin structure (tightly packed versus loosely packed) and gene expression

• Chemical modification of histones include acetylation and methylation

• Chemical modification of DNA include methylation

Histone Modifications

• In histone acetylation, acetyl groups (-COCH3)are attached to positively charged lysines in histone tails

• This process loosens chromatin structure, thereby promoting the initiation of transcription

Fig. 18-7

Histonetails

DNAdouble helix

(a) Histone tails protrude outward from a nucleosome

Acetylated histones

Aminoacidsavailablefor chemicalmodification

(b) Acetylation of histone tails promotes loose chromatin structure that permits transcription

Unacetylated histones

DNA Methylation

• DNA methylation, the addition of methyl groups (CH3) to certain bases in DNA, usually cytosine, is associated with reduced transcription in some species

• DNA methylation can cause long-term inactivation of genes in cellular differentiation

• Barr Bodies• Once methylated, genes usually remain so through

successive cell divisions• After replication, enzymes methylate the correct daughter

strand so that the methylation pattern is inherited

Epigenetic Inheritance

• Though chromatin modifications do not alter DNA sequence, they may be passed to future generations of cells

• The inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence is called epigenetic inheritance

• Epigenetic modifications can be reversed, unlike mutations in DNA sequence

Epigenetic Inheritance

• Might explain why one twin gets a genetic disease (schizophrenia) and the other does not.

• Alterations in normal DNA methylation patterns are seen in some cancers-these changes can cause inappropriate gene expression in cancer cells.

Regulation of Transcription Initiation

• Chromatin-modifying enzymes provide initial control of gene expression by making a region of DNA either more or less able to bind the transcription machinery

• Regulation of transcription by enhancers (and their associated control elements) is another important mechanism by which initiation of transcription can be regulated.

Organization of a Typical Eukaryotic Gene

• Associated with most eukaryotic genes are control elements, segments of noncoding DNA that help regulate transcription by binding certain proteins

• Control elements and the proteins they bind are critical to the precise regulation of gene expression in different cell types

Figure 15.8

DNAUpstream

Enhancer(distal control

elements)

Proximalcontrol

elementsTranscription

start site

Exon Intron Exon

Promoter

Intron Exon

Poly-A signalsequence

Transcriptiontermination

region

Down-stream

Transcription

Exon Intron IntronExon Exon

Poly-Asignal

Primary RNAtranscript(pre-mRNA)

5 Cleaved 3end ofprimarytranscript

Intron RNA

mRNA

RNA processing

Coding segment

3

5 5 3Cap UTRStart

codonStop

codon UTR Poly-Atail

G P P P AAAAAA

The Roles of Transcription Factors

• To initiate transcription, eukaryotic RNA polymerase requires the assistance of proteins called transcription factors. React with proximal control elements of the gene (DNA)

• General transcription factors are essential for the transcription of all protein-coding genes

• If a gene is going to be transcribed at anything but a slow rate, specific transcription factor must also be involved.

• In eukaryotes, high levels of transcription of particular genes depend on control elements interacting with specific transcription factors

• An activator is a protein (a specific transcription factor) that binds to a (distal) control element of an enhancer and stimulates transcription of a gene

• Activators have two domains, one that binds DNA and a second that activates transcription

• Bound activators cause mediator proteins to interact with proteins at the promoter

Enhancers and Specific Transcription Factors

Figure 15.9

Activationdomain

DNA

DNA-bindingdomain

• Bound activators are brought into contact with a group of mediator proteins through DNA bending

• The mediator proteins in turn interact with proteins at the promoter

• These protein-protein interactions help to assemble and position the initiation complex on the promoter

Figure 15.10-3

DNA

EnhancerDistal controlelement

Activators PromoterGene

TATA box

DNA-bendingprotein

Group of mediator proteins

General transcriptionfactors

RNApolymerase II

RNApolymerase II

RNA synthesisTranscriptioninitiation complex

Why does a liver cell produce albumin and a lens cell crystalline protein?

• All activators needed for high-level expression of the albumin gene are present in liver cells but not lens cells.

• All activators needed for high-level expression of the crystalline gene are present in lens cells but not liver cells.

• The presence of activators in cells may occur at precise times during development or in a particular cell type

Figure 15.11Albumin gene

Crystallin gene

Promoter

Promoter

(b) LENS CELL NUCLEUS

Availableactivators

Albumin genenot expressed

Crystallin geneexpressed

Crystallin genenot expressed

Albumin geneexpressed

Availableactivators

(a) LIVER CELL NUCLEUS

Controlelements

Enhancer

Enhancer

Mechanisms of Post-Transcriptional Regulation

• Transcription alone does not account for gene expression

• Regulatory mechanisms can operate at various stages after transcription

• Such mechanisms allow a cell to fine-tune gene expression rapidly in response to environmental changes

Figure 15.UN02

Chromatin modification

Transcription

RNA processing

TranslationmRNAdegradation

Proteinprocessing

and degradation

RNA Processing

• In alternative RNA splicing, different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns

• In Drosophila-one gene has enough exons that alternative splicing can produce 19,000 different membrane proteins. Each nerve cell has a different membrane protein. Estimates are that 90% of human genes are alternatively spliced.

Figure 15.12

DNA

PrimaryRNAtranscript

mRNA or

Exons

Troponin T gene

RNA splicing

1 2 3 4 5

1 2 3 5 1 2 4 5

1 2 3 4 5

Figure 15.UN03

Chromatin modification

Transcription

RNA processing

TranslationmRNAdegradation

Proteinprocessing

and degradation

mRNA Degradation

• The life span of mRNA molecules in the cytoplasm is important in determining the pattern of protein synthesis in a cell

• Eukaryotic mRNA generally survives longer than prokaryotic mRNA

• Nucleotide sequences that influence the life span of mRNA in eukaryotes reside in the untranslated region (UTR) at the 3 end of the molecule

• Noncoding rna’s can cause m-rna to be degraded or have it inhibited (more about that later)

Protein Processing and Degradation

• After translation, various types of protein processing, including cleavage and chemical modification, are subject to control (pro-insulin insulin)

• The length of time each protein functions in a cell is regulated by means of selective degradation.To mark a particular protein for destruction, the cell commonly attaches molecules of ubiquitin to the protein, which triggers its destruction

Figure 15.UN06Chromatin modification Transcription

RNA processing

Translation

mRNA degradation Protein processing and degradation

• Protein processing and degradation aresubject to regulation.

• Each mRNA has acharacteristic life span.

• Initiation of translation can be controlledvia regulation of initiation factors.

mRNA or

Primary RNAtranscript

• Alternative RNA splicing:

• The genes in a coordinately controlledgroup all share a combination of controlelements.

• Regulation of transcription initiation:DNA control elements in enhancers bindspecific transcription factors.

Bending ofthe DNAenablesactivators tocontact proteins atthe promoter, initiating transcription.

• Genes in highly compactedchromatin are generally nottranscribed.• Histone acetylation seemsto loosen chromatinstructure,enhancingtranscription.

• DNA methylation generallyreduces transcription.

Chromatin modification

Transcription

RNA processing

mRNAdegradation

Translation

Protein processingand degradation

Noncoding RNAs play multiple roles in controlling gene expression

• Protein coding DNA only accounts for about 1.5% of the human genome

• Only a small fraction of DNA codes for rRNA, and tRNA. What is the rest of DNA for?

• A significant amount of the genome may be transcribed into noncoding (nc)RNAs (rock star versus backups)

• Noncoding RNAs regulate gene expression at the level of translation by degrading M-rna or blocking its translation

Effects on mRNAs by MicroRNAs and Small Interfering RNAs

• MicroRNAs (miRNAs) are small single-stranded RNA molecules that can bind to complementary mRNA sequences

• These can degrade the mRNA or block its translation

Fig. 18-13

miRNA-proteincomplex(a) Primary miRNA transcript

Translation blocked

Hydrogenbond

(b) Generation and function of miRNAs

Hairpin miRNA

miRNA

Dicer

3

mRNA degraded

5

• Another class of small RNAs are called small interfering RNAs (siRNAs)

• siRNAs and miRNAs are similar but form from different RNA precursors

• The phenomenon of inhibition of gene expression by siRNAs is called RNA interference (RNAi)

• RNAi may represent the evolution of a genetic control system from a defense mechanism against double stranded RNA virus infection.

• Researchers uses RNAi to knock out genes and study their function.

Researchers can monitor expression of specific genes

• Cells of a given multicellular organism differ from each other because they express different genes from an identical genome

• The most straightforward way to discover which genes are expressed by cells of interest is to identify the mRNAs being made

Studying the Expression of Single Genes

• We can detect mRNA in a cell using nucleic acid hybridization, the base pairing of a strand of nucleic acid to its complementary sequence

• The complementary molecule in this case is a short single-stranded DNA or RNA called a nucleic acid probe

• Each probe is labeled with a fluorescent tag to allow visualization

• The technique allows us to see the mRNA in place (in situ) in the intact organism and is thus called in situ hybridization

Figure 15.14

50 m

• Genome-wide expression studies can be carried out using DNA microarray assays

• A microarray—also called a DNA chip—contains tiny amounts of many single-stranded DNA fragments affixed to the slide in a grid

• The experiment can identify subsets of genes that are being expressed differently in one sample (breast cancer tissue for example)compared to another (normal tissue).

Figure 15.17

Genes in redwells expressedin first tissue.

Genes in greenwells expressedin second tissue.

Genes in yellowwells expressedin both tissues.

Genes in blackwells notexpressed in either tissue.

DNA microarray

Notes for microarray diagram

• Each little dot represent a well in a plate.• Each well has a gene from the two tissues

tested. This plate has 5,760 wells (about 25% of human genes).

• The plate is incubated with probes from the two tissues (one tissues probes are labeled green and the other red).

• Colors for each well indicate whether the gene is expressed in one tissue (red or green), both tissues (yellow) or neither (black)