15 regulation of gene expression expression of genetic information is dependent on regulatory...

15 Regulation of Gene Expression

Expression of genetic information is dependent on regulatory mechanisms that either activate or repress the transcription of genes. Transcription is modulated by theinteraction at various regulatory molecules with DNA sequences, most often located upstream from affected genes.Genetic regulation in eukaryotes also occurs during post transcriptional events.

Gene expression• Transcription

– Initiation Elongation Termination

Francois Jacob

•Translation–Initiation

–Elongation

–Termination

Jacques Monod

• 15.1 Genetic Regulation in Prokaryotes:An Overview15.2 Lactose Metabolism in E. coli: An Inducible SystemStructural GenesThe Discovery of Regulatory MutationsThe Operon Model: Negative'ControlGenetic Proof of the Qperon ModelIsolation of the lac RepresserThe CAP Protein: Positive Control of the lac Operon

• 15.3 Tryptophan Metabolism in E. coli:A Repressible Gene SystemGenetic Evidence for the trp Operon ,Attenuation15.4 Genetic Regulation in Eukaryotes:An Overview

• 15.5 Regulatory Elements, Transcription Factors, and Eukaryotic GenesPromotersEnhancersTranscription FactorsStructural Motifs of Transcription FactorsAssembly of the Transcription ComplexChromatin Conformation, DNA Methylation,and Gene Expression15.6 Gene Regulation by Steroid Hormones15.7 Posttranscriptional Regulation of GeneExpression:Alternative Splicing of mRNA

15.1 Genetic Regulation in Prokaryotes:An Overview

• Regulation of gene expression has been studied extensively in prokaryotes, particularly in Escherichia coll. Highly efficient mechanisms have evolved that turn genes on and off, depending on the cell's metabolic needs in particular environments. Detailed analysis of proteins in E. coll has shown that for the more than 4000 polypeptide chains encoded by the genome, a vast range of concentration of gene products exists. Some proteins may be present in as few as 5-10 molecules per cell, whereas others, such as ribosomal proteins and the many proteins involved in the glycolytic pathway, are present in as many as 100,000 copies per cell. The idea that microorganisms regulate the synthesis of gene products can be illustrated by considering the utilization of lactose (a galactose-glucose-containing disaccha-ride) as a carbon source. When it is present in the growth medium, many bacteria and yeast produce enzymes specific to lactose metabolism. When lactose is absent, the enzymes are not manufactured. These organisms thus "adapt" to their environment, producing certain enzymes only when specific chemical substrates are present. Such enzymes are said to be inducible, reflecting the role of the substrate, which serves as the inducer of enzyme production. In contrast, other enzymes that are produced continuously, re-gardless of the chemical makeup of the environment, are described as constitutive.Studies have also revealed cases where the presence of a specific molecule causes inhibition of genetic expression. This is often the case for molecules that are the end products of biosynthetic pathways. Amino acids can be synthesized by bacterial cells, but if the amino acids are present in the growth medium, they can be taken up and used. In such cases, it is inefficient for the cell to produce the enzymes necessary for the synthesis of those amino acids, and transcription of mRNA for the appropriate biosynthetic enzymes is repressed. This is an example of a repressible system of gene regulation.Regulation, whether it is inducible or repressible, may be under either negative or positive control. Under negative control, genetic expression occurs unless it is shut off by some form of a regulator molecule. In contrast, under positive control, transcription occurs only if a regulator molecule directly stimulates RNA production. In theory, either type of control can govern inducible or repressible systems. Our discussion in the ensuing sections of this chapter will clarify these contrasting systems of regulation. For the enzymes involved in lactose and tryptophan, negative control is operative.

Regulation of Gene Expression

• At Transcription stage:– Economical, but can’t be reversed quickly

• At Translation stage:– Wasteful, but easily reversible

Feedback regulation:

– Positive control: the gene is off unless it is turned on

– Negative control: the gene is on unless it is turned off

Feedback regulation:

– Induction: the “target” molecule turns expression ON (e.g., by disactivating the repressor)

– Repression: the “target” molecule turns expression OFF

The lac Operon in E. coli

Genetic System under both Induction and Repression regulation.

Let’s first consider Induction.

Cis- and trans- acting regulatory sequences

• Operator: a cis-acting elementInfluences expression of genes downstream

from it on the same 2-stranded DNA

• Repressor gene: a trans- acting regulatory geneInfluences expression of any relevant genes

in the same cell, on the same or different DNA

Let’s stop here and solve some problems

• You have 2 constitutive lac mutants (always expressing lac operon). You transform them with a plasmid containing an intact lac operon. One of them changes the phenotype to wild type. The other does not no matter how much you try. What may be the genotypes of these 2 mutants?

Let’s stop here and solve some problems

• You have a mutant strain with a mutation in lac operon repressor gene, which prevents lactose from binding to repressor protein.

• What will be the phenotype? Can it be changed by transforming this strain with a plasmid containing any lac operon structural or regulatory genes?

Homework!

• Textbook, Ch. 17, p.459: #5, 6, 10(extra spicy, optional)

• Problem manual, p.82-83: #44, 48 (solved)

Pop-up quiz:

Genotype Phenotype

(lactose present)

Phenotype

(lactose absent)

I- P+ O+ Z+(I- codes for inactive repressor protein)

I- P+ O+ Z+/

I+ P+ O+ Z-

CAP-cAMP control

Glucose present ->

cAMP level decreases ->

no CAP-cAMP complex is formed ->

no CAP binding to promoter ->

no activation of transcription

Let’s compare two controls of lac operon

• Repressor-operator control

• CAP-cAMP control

Let’s compare two controls of lac operon

• Repressor-operator control

Negative control

Control molecule: lactose

• CAP-cAMP control

Positive control

Control molecule: glucose

Another operon under double control:

Trp operon

Trp = triptophan

Let’s compare regulation of of lac operon and trp operons

• lac operon

Induction (target molecule, lactose, induces expression by desactivating repressor protein)

• trp operon

Repression (target molecule, triptophan, represses expression by activating repressor protein)

But.. It’s not the whole story!

Attenuation regulation of trp-operon

Regulation of Gene Expression in Prokaryotes

Hartwell pp. 551-560, 567-571

Regulation of transcription

• Differences in the basepairs in the -35 and -10 boxes allow genes to be expressed at different levels.

• This kind of regulation is important, but it does not allow the cell to adjust its pattern of gene expression in a dynamic manner to meet changing needs and environmental stresses.

Strategies for regulating gene expression in prokaryotes

• Switches in the subunit of RNA polymerase

• Control by a regulated repressor of transcription

• Control by a regulated activator of transcription

• Regulated attenuation (termination) of transcripts

Switches in the subunit of RNA polymerase

• Under certain conditions, the cell needs to induce a large set of genes that are normally silent

• Examples include:– heat-shock proteins– nitrogen starvation– developmental changes such as sporulation

Switches in the subunit of RNA polymerase

• Under certain conditions, the cell needs to induce a large set of genes that are normally silent

• This can be accomplished by synthesizing or activating a different RNA polymerase subunit that recognizes a distinct set of promoter sequences.

70 -32 -

pg. 786

Strategies for regulating expression in prokaryotes

In prokaryotes, genes with related functions can be expressed as a single mRNA controlled by one promoter

O LacY LacA

lactose permease-galactosidase unknown function

Promoter

Transcription

The lac operon

Operons are regulated by two kinds of elements

Operator

Transcription

Promoter Gene 1

RNAP Transcriptional regulatory proteins

Regulatory sites

Transcriptional regulatory proteins

• Often called transcription factors• Some transcriptional regulatory proteins

stimulate transcription initiation (called activators)

• Other transcriptional regulatory factors inhibit transcription (called repressors)

• In bacteria, the sites where transcription factors bind reside close to the transcription start site. These binding sequences are called operators.

The regulatory gene for an operon can reside at another site of the chromo

pg. 869

The Lac Operon:A classic example of dynamic regulation of g

ene expression

• -galactosidase

pg. 868

The addition of lactose to E. coli leads to a large increase in -galactosidase synthesis

-gal synthesis is approximately6% of total protein synthesis

-gal synthesis stops

(cell growth)

- -galactosidase expression is inducible.

pg. 869

The addition of lactose to E. coli leads to a large increase in -galactosidase synthesis

-gal synthesis is approximately6% of total protein synthesis

-gal synthesis stops

(cell growth)

-Note: The increase in -galactosidase synthesis only occurs in the absence of preferred carbon/energy sources such as glucose.

pg. 869

Isolation of mutations affecting the induction of the lac operon

Inducers of the lac operon

1,6-Allolactose

-the natural inducer of lac operon expression-formed from lactose by an activity of -galactosidase

-a synthetic inducer of lac operon expression-cannot be metabolized

Assaying for -galactosidase activity using X-Gal substrate

wild-type E. coli grown on mediawith X-gal but without glucose

wild-type E. coli grown on mediawith X-gal and IPTG but without glucose

mutagenize

plate on mediawith X-gal and

lacking IPTG andglucose

a mutant that expresses-galactosidase without inducer

Constitutive mutant

mutagenize

plate on mediawith IPTG, X-gal

and lacking glucose

a mutant that fails to expressfunctional -galactosidase

• Most of these mutation have inactivated the lacZ gene rather than affected its expression.

• However, there are mutants in which the expression of lacY and lacA have also been lost.

Uninducible mutant

Production of B-galactosidase by strains grown under different conditions

IPTG - - + +

Glucose - + - +

Strain:

WildType no no yes no

LacZ- no no no no

oc yes no yes no

i- yes no yes no

is no no no no

Genetic analysis of lac operon regulation identifies two key components

• Transcriptional regulatory protein– encoded by the lacI gene (maps near the lac operon)

– loss-of-function lacI (i-) mutations cause the lac operon to be constitutively expressed

• This indicates that LacI acts as a repressor of lac operon transcription in the absence of inducer

• Site for lac repressor binding– called the lac operator (lacO)

– mutations of the lac operator (oc) also lead to failure of lac repressor binding and constitutive expression

Regulation of the lac operon

pg. 872

Using bacterial merodiploids to perform complementation tests

• E. coli are haploid,

• but some E. coli have large plasmids called F factors that can be transmitted (mated) from one E. coli to another.

• Through complicated genetic tricks, it is possible to isolate strains in which the F factor contains a large region of E. coli chromosome including the lac operon.

• Such a partially diploid strain is called merodiploid.

F factor

lac lac

E. coli chromosome

Transcriptional regulatory protein

Operator

Transcription

Promoter Allele 2

Operator

Transcription

Promoter Allele 1

Cis vs. Trans

• Trans-acting: In a cell with two copies of an operon, the transcriptional regulatory protein will regulate the expression of either allele,

• Cis-acting: An operator (the site where a transcriptional regulatory protein binds) can only affect the allele to which it is joined.

F factor

lacI-, lacO+, lacZ+ lacI+, lacO+, lacZ-

Evidence that lacI encodes a repressor: the LacI protein can act in trans

• Result: Inducible synthesis

Constitutive Uninducible

Evidence that lacO acts in cis

F factor

lacI+, lacO-, lacZ- lacI+, lacO+, lacZ+

F factor

lacI+, lacOc, lacZ+ lacI+, lacO+, lacZ-

inducible

constitutive

• Dominant mutations that gave uninducible lac operon expression were also isolated in the lacI gene.• These encode a “super repressor” (is) that cannot bind the inducer and therefore is always bound to t

he operator

Biochemical analysis of LacI repressor action

• Identification of the lac operator sequence

Biochemical confirmation that LacI repressor acts by binding to the lac operator in the absence of the inducer

QuickTime™ and aPhoto - JPEG decompressor

are needed to see this picture.

Hartwell, pg. 540

Lac repressor binds the operator as a dimer

The lac repressor binding site overlaps the RNA polymerase and tran

scription initiation sites

Hartwell, pg. 543

Summary of analysis of LacI repressor action

• Results– Lac repressor binds the operator region.

– Constitutive mutants • change the operator so that it cannot bind repressor (oc)

• change the repressor so that it cannot bind the operator (i-)

– The addition of inducer prevents lac repressor from binding the operator.

– Uninducible mutants (is) change the repressor so that it cannot bind inducer (and therefore stays bound to the operator)

Strategies for regulating expression in prokaryotes

Catabolite Activator Protein

• Even in the presence of lactose, the lac operon is poorly expressed if a preferred carbon/energy source such as glucose is present.

• The lac operon promoter is very weak and requires another transcriptional regulatory protein to be strongly expressed.

• This protein is called catabolite activator protein (CAP).

O LacY LacAP LacZ

Transcription

Catabolite activator protein• E. coli has a system for monitoring its energy levels.• When energy stores are low, the enzyme adenylcycl

ase catalyzes the synthesis of cyclic AMP from ATP.

• cAMP serves as a signal to turn on operons responsible for digesting (catabolizing) less-preferred sugars such as lactose.

Catabolite activator protein

Transcriptional activator

Catabolite activator protein

• CAP-cAMP binds to the promoter next to the RNA polymerase binding site.

• CAP makes contacts with RNA polymerase that increase the binding of RNA polymerase to the lac promoter.

Hartwell, pg. 543

lactose and glucose

no lactose or glucose

lactose without glucose

glucose without lactose

The trp operon: a classic model for attenuation

• The trp operon encodes the enzymes required to synthesize tryptophan (TrpA-E).

• Synthesis of the trp mRNA is controlled by a repressor that blocks transcription when bound to tryptophan.

• Therefore, when tryptophan levels are high, transcription will be repressed.

The trp operon

Hartwell, p. 546

The Trp operon is regulated by a repressor

Hartwell, p. 546

• However, in the absence of trp repressor, the synthesis of trp mRNA is still partially repressed by the presence of tryptophan in the growth medium.

Hartwell, p. 546

Attentuation: The trp operon mRNA contains a leader region that can fold into different stem-loop structures

Trp codons in the leader sequence sense the availability of tryptophan

• The trp mRNA leader contains two trp codons in a row.

• When the tryptophan supply in the cell is limited, ribosomes will stall at these trp codons.

• The ability of the ribosome to read through these codons regulates the stem-loop choice.

The availability of tryptophan regulates which stem loop structure forms

Other operons encoding amino acid biosynthesis enzymes are also regulate

d by attentuation

p. 888

Regulation of Gene Expression in Eukaryotes

Hartwell pp. 250-256, 581-590, 602-609

Transcription and translation in prokaryotes vs eukaryotes

• In prokaryotes, transcription and translation are tightly coupled.

• In contrast, transcription and translation are spatially separated in eukaryotes.

Primary RNA transcripts are extensively modified before leaving the nucleus

• Nascent mRNA is “capped” with a 7-methylguanosine at the 5’ end

• 3’ end is polyadenylated (100-200 residues) to protect against degradation

• mRNA editing can occur in rare cases• Splicing: often introns must be removed from tra

nscript and protein-coding exons spliced together to form mature mRNA for translation

• 5’ capping– protects mRNA from ribonucleases– promotes translation– Capping enzyme adds backward G– Then methyl transferases modify G

and 1 or more nucleotidesFirst base of transcript

• 3’ polyadenylation– Cleavage and polyA additio

n are initiated via recognition of AAUAAA site 11-30 nucleotides upstream of tail

– Protects RNA from rapid degradation

– May aid transport out of nucleus

– May increase efficiency of translation

Primary RNA transcripts are extensively modified before leaving the nucleus• mRNA editing

– only a few examples are known

synthesized in the intestine

deaminase is onlypresent in small intestine

synthesized in the liver

• mRNA splicing• Some parts of the RNA t

ranscript are removed prior to translation

• the excised pieces are called introns (intervening)

• the remaining pieces are called exons (expressed)

The mechanism of RNA splicing

• Certain sequences are conserved near splicing junctions and serve as splicing signals

• Some key bases are in the intron and others are in the flanking exons

The mechanism of splicing

• Splicing involves the excision of the intron in a lariat form.

• Splicesomes are large RNA- and protein-containing complexes.

• Some of the RNAs are used to recognize the splice junctions.

• Example: U1 snRNA recognizes 5’ splice site

• Splicesomes are large RNA- and protein-containing complexes

• Some of the RNAs are used to recognize the splice junctions

• Example: U2 snRNA recognizes branch site

Spliceosome Assembly

Why splicing?

• mRNA splicing can allow a single gene to express several different proteins (isoforms)

• These different forms often have different activities, can interact specifically with different molecules– For example, splicing can result in secreted vs membrane

bound forms of a protein like an antibody

• The production of different isoforms can be regulated during development– The Drosophila sex determination system provides several

classic examples of developmentally regulated alternative splicing giving rise to differing functions

Splicing can produce a secreted vs membrane-bound antibody

• Alternative splicing of this heavy chain gene can produce two different types of antibody with the same specificity

• When activated by antigen, B-cells begin secreting their antibodies

• Secreted form lacks exons 7 and 8 which encode membrane attachment domain

2 63 541

Translation startStop Stop

Doublesex female isoform

Doublesex male isoform

activates female-specific genesand represses male-specific genes

activates male-specific genesand represses female-specific genes

Drosophila Doublesex is alternatively spliced into male or female forms

Functional Transformer

No functional Transformer

Regulation of transcriptional initiation in eukaryotes

• Three different RNA polymerases transcribe genes in eukaryotes– RNA polymerase I transcribes rRNAs (18S, 5.8

S and 28S)– RNA polymerase II transcribes mRNAs and sn

RNAs– RNA polymerase III transcribes tRNAs and 5s r

• Each polymerase consists of many subunits.

Promoter elements in prokaryotes

• In prokaryotes, the promoter elements are well-defined.

• They are always present and always in the same position relative to the start site of transcription.

Promoter elements in eukaryotes

• In eukaryotes, the promoter elements are much less well-defined.

• They are not always present and can be various distances from the start site of transcription.

usually present

frequently not present

Transcription factors TFIIA, B, D, E and F play important roles in transcript in

itiation by RNA polymerase II

• These factors were identified by purifying the proteins required for correct initiation in vitro.

• Each factor is composed of multiple proteins.• TFIID contains the 30 kilodalton (kD) TATA-box

binding protein.• This complex of proteins together is called the bas

al transcriptional machinery.

Sequentialassemblyof thebasaltranscriptionmachinery

Enhancer elements control the activity of eukaryotic promoters

• Eukaryotic promoters are invariably weak and require “enhancer elements” to drive high level expression.

• Enhancer elements are defined as DNA sequences that promote the expression of a gene.– Enhancer elements are distinguished from promoters in that t

heir function does not depend on their orientation relative to the transcription units.

(TATA)

Enhancer

(TATA)

Enhancer

Enhancer elements control the activity of eukaryotic promoters

• Enhancer elements do not need to be placed at a precise distance from the promoter.

• Enhancer elements are frequently tens of kilobasepairs (Kb) away from the transcriptional start site.

(TATA)

Enhancer

(TATA)

Enhancer

Enhancer elements contain multiple binding sites for transcriptional activator proteins

• Many developmentally important genes have enhancers with binding sites for many different transcriptional activator proteins.

• Transcriptional activator proteins frequently consist of two modular domains:

• Many transcriptional activators have negatively-charged regions that may interact with components of the basal transcription machinery.

• Removing the activation domain can convert a transcriptional activator into a repressor

DNA looping allows transcriptional activator proteins to interact with the TFII components

• Silencer elements that provide binding sites for transcriptional repressor proteins also exist

Repressor proteins reduce transcription through competition or quenching

• Repressor can compete with activator for enhancer site

• Repressor can quench activator by– Blocking DNA-binding

domain– Blocking activation

domain

The expression pattern of an individual gene is determined by the transcription factors th

at bind its enhancers and their regulation

How is the activity of enhancers regulated during development and in respo

nse to environmental changes?• Many types of regulation are observed:

– tissue-specific expression• Many transcriptional activators are only expressed in specific cell types.

– by binding to hormones• For example, steroid receptors are enhancer binding proteins whose activity d

epends on hormone binding.

– by phosphorylation• Many transcription factors are only active when phosphorylated. These inclu

de transcription factors that respond to hormones and growth factors such as insulin.

• Together, these kinds of regulation can limit gene expression to specific times and tissues.

The GAL4, GAL80 Gene System

• One enhancer controls the genes GAL1,7, and 10

• Its activator, GAL4, is quenched by GAL80 in absence of galactose

• GAL1 and GAL3 can be induced by galactose to bind GAL80 and prevent it from blocking GAL4

• This allows GAL1,7, and 10 to be transcribed at high levels

• Heat Shock(HS) driven Flp enzyme causes recombination between homologous FRT sites during mitosis

• This derepresses a tissue-specific Gal4 transgene by recombining away Gal80 repressor

• Leading to clones of cells positively marked by the membrane marker CD8-GFP

Lee and Luo, Neuron 1999

Mosaic Analysis with a Repressible Cell MarkerMARCM

nc82CD8-GFP

MARCM can label a single neuron in the fly brain

Two types of regulation that we will not have time to discuss

• Regulatory proteins that act by modifying chromatin structure to allow transcription– Nucleosomes block the promoters of most inactive genes.

– Some proteins such as the SWI-SWF complex in yeast act by modifying chromatin structure to give the basal transcription machinery greater access to promoters.

• Regulation of mRNA accumulation by controlling mRNA stability– Length of poly-A tail

– RNA-binding proteins that protect RNA from degradation

Regulation of Gene Expression in Eukaryotes

Chapter 21

Eukaryotic gene regulation is different from regulation of prokaryotic genes

• Complex chromosome structure• Multiple chromosomes• Spatial & temporal separation of

transcription and translation• Transcripts processed in nucleus• mRNA lasts longer• Must have translational controls• Multicellular with many cell typ

Eukaryotic genes have promoters and enhancers

promoterenhancer

Eukaryotic genes vary in the number and arrangement of controlling elements

Mutations in the Promoter Region Can Drastically Alter Transcription

Overview of initiation of eukaryotic transcription

•TBP of TFIID binds ~20bp•IIA & IIB Bind•RNA Polymerase II, IIF binds•IIE,IIH, IIJ bind•Promoter clearance occurs•basal transcription ensues

Enhancers control chromatin structure and transcription rate

•Different from promoters because….•Position need not be fixed•Orientation is not critical•Introduced enhancers speed up transcription

How do they work?

Enhancers cause loops in the DNA which allow TFs to interact with transcription complex

Stabilizes transcription complex

Helix-turn-helix

Transcription factors have functional domains that permit binding to DNA and others that facilitate binding to protein in transcription complex

True activator transcription factors include….

Zinc Fingers

and…..

Leucine Zippers

and finally…...

Antirepressor Transcription factors

• Change chromatin structure to allow polymerase binding

•Several processes involved

ATP dependent remodeling (SWI/SNF)

Histone modification by Histone Acetyltransferase Enzymes (HAT)

•Acetylation of basic amino acids of histone lessen interaction with DNA•Deacetylases (HD) reverse this

Methylation of CG doublets regulates gene expression•Expression high when methylation low•tissue specific

Alternative splicing of mRNA occurs in different tissues•E.g. preprotachykin gene product

Controlling mRNA stability

• General or cell specific

• Stability sequences

• Instability sequences (e.g. AUUUA on fos mRNA)

• Address sequences

• Translational controls

Translation controls can be used to regulate final gene products

•Tubulin gene regulated by tubulin subunit conc. in cytoplasm (autoregulation)

15 regulation of gene expression expression of genetic information is dependent on regulatory...

gene regulation

genetic regulation

transcription of genes

synthesis of gene products

enzymes specific

lactose metabolism

negativecontrol genetic

regulatory mechanisms

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