Gene Expression in Gene Expression in ProkaryotesProkaryotes

Why regulate gene Why regulate gene expression?expression?

It takes a lot of energy to make RNA and It takes a lot of energy to make RNA and protein.protein.

Therefore some genes active all the time Therefore some genes active all the time because their products are in constant because their products are in constant demand.demand.

Others are turned off most of the time Others are turned off most of the time and are only switched on when their and are only switched on when their products are needed.products are needed.

The control of gene expression is much more The control of gene expression is much more complex in eukaryotes than in prokaryotes.complex in eukaryotes than in prokaryotes.

Reasons being, Eukaryotes have:Reasons being, Eukaryotes have:

– Compartmentalization of cellsCompartmentalization of cells

– More extensive transcript processing More extensive transcript processing

– Regulation from a distance Regulation from a distance

– Cell and tissue specific gene expressionCell and tissue specific gene expression

– Larger Genome sizeLarger Genome size

– Genes scattered about the genomeGenes scattered about the genome

In prokaryotes, the control of transcriptional In prokaryotes, the control of transcriptional initiation is the major point of regulationinitiation is the major point of regulation

In eukaryotes the regulation of gene expression is In eukaryotes the regulation of gene expression is controlled nearly equivalently from many different controlled nearly equivalently from many different points:points:

– Initiation of transcription (most important control)Initiation of transcription (most important control)– Chromatin controlChromatin control– Epigenetic controlEpigenetic control– Transcript processingTranscript processing– Transcript stabilityTranscript stability– RNA transportRNA transport– Protein stabilityProtein stability– Protein transportProtein transport– Post-Translational modificationsPost-Translational modifications

Gene Control in ProkaryotesGene Control in Prokaryotes

One way in which prokaryotes control One way in which prokaryotes control gene expression is to group gene expression is to group functionally related genes together so functionally related genes together so that they can be regulated together.that they can be regulated together.

This grouping is called an operon.This grouping is called an operon.

The clustered genes are transcribed The clustered genes are transcribed together from one promoter giving a together from one promoter giving a polycistronic messenger.polycistronic messenger.

Gene Control in ProkaryotesGene Control in Prokaryotes

An operon can be defined as a cluster An operon can be defined as a cluster gene that encode the proteins necessary to gene that encode the proteins necessary to perform coordinated functionperform coordinated function

Genes of the same operon have related Genes of the same operon have related functions within the cell and are turned on functions within the cell and are turned on (expressed) and off together (suppressed).(expressed) and off together (suppressed).

The first operon discovered was the The first operon discovered was the lac lac operonoperon so named because its products are so named because its products are involved in lactose breakdown.involved in lactose breakdown.

An operon consists of:An operon consists of:

– a a promoterpromoter (binding site for RNA (binding site for RNA polymerase)polymerase)

– a repressor binding site called an a repressor binding site called an operator operator that overlaps the promoter.that overlaps the promoter.

– structural genesstructural genes

OperatorOperator Repressor proteins encoded by repressor Repressor proteins encoded by repressor

genes, are synthesized to regulate gene genes, are synthesized to regulate gene expression.expression.

They bind to the operator site to block They bind to the operator site to block transcription by RNA polymerase. transcription by RNA polymerase.

PromoterPromoter The promoter sequences are recognized by The promoter sequences are recognized by

RNA polymerase. RNA polymerase.

When RNA polymerase binds to the promoter, When RNA polymerase binds to the promoter, transcription occurstranscription occurs

ActvatorsActvators The activity of RNA polymerase is also The activity of RNA polymerase is also

regulated by interaction with regulated by interaction with accessory proteins called activatorsaccessory proteins called activators

The presence of the activator The presence of the activator removes repression and transcription removes repression and transcription occursoccurs

Two major modes of transcriptional regulation Two major modes of transcriptional regulation function in bacteria (function in bacteria (E. coliE. coli) to control the ) to control the expression of operons: expression of operons:

– repression and repression and

– induction.induction.

Both mechanisms involve repressor proteins. Both mechanisms involve repressor proteins.

InductionInduction happens in operons that produce gene happens in operons that produce gene products needed for the utilization of energy.products needed for the utilization of energy.

RepressionRepression regulates operons that produce gene regulates operons that produce gene products necessary for the synthesis of small products necessary for the synthesis of small biomolecules such as amino acids. biomolecules such as amino acids.

Inducible systemInducible system Negative controlNegative control

the effector molecule interacts with the repressor the effector molecule interacts with the repressor protein such that it cannot bind to the operator protein such that it cannot bind to the operator

With inducible systems, the binding of the effector With inducible systems, the binding of the effector molecule to the repressor:molecule to the repressor:

– greatly reduces the affinity of the repressor for greatly reduces the affinity of the repressor for the operatorthe operator

– the repressor is released and transcription the repressor is released and transcription proceeds. proceeds.

In addition to negative control mediated In addition to negative control mediated by a repressor, expression from an by a repressor, expression from an inducible operon is also under positive inducible operon is also under positive control, mediated by an activatorcontrol, mediated by an activator

A classic example of an inducible (catabolite-A classic example of an inducible (catabolite-mediated) operon is the mediated) operon is the lac operonlac operon, , responsible for obtaining energy from responsible for obtaining energy from galactosides such as lactose. galactosides such as lactose.

Repressible systemRepressible system

Negative controlNegative control

the effector molecule interacts with the repressor the effector molecule interacts with the repressor protein such that it can bind to the operator protein such that it can bind to the operator

With repressible systems, the binding of the With repressible systems, the binding of the effector molecule to the repressor:effector molecule to the repressor:

– greatly increases the affinity of repressor for greatly increases the affinity of repressor for the operator the operator

– the repressor binds and stops transcription. the repressor binds and stops transcription.

For the For the trptrp operon , the addition of tryptophan operon , the addition of tryptophan (the effector molecule) to the (the effector molecule) to the E. coli E. coli environment environment shuts off the system because the repressors binds shuts off the system because the repressors binds at the operator.at the operator.

In addition to negative control mediated by a In addition to negative control mediated by a repressor, expression from a repressible repressor, expression from a repressible operons is attenuated by sequences within operons is attenuated by sequences within the transcribed RNA.the transcribed RNA.

A classic example of a repressible (and A classic example of a repressible (and attenuated) operon is the attenuated) operon is the trp operontrp operon, responsible, responsible

for the biosynthesis of tryptophan. for the biosynthesis of tryptophan.

Structure of the Structure of the laclac Operon Operon

The The laclac operon three structural genes: operon three structural genes:

– ZZ– yy – aa

The The zz gene codes for gene codes for ββ-galactosidase , responsible -galactosidase , responsible for the hydrolysis of the disaccharide, lactose into for the hydrolysis of the disaccharide, lactose into its monomeric units, galactose and glucose. its monomeric units, galactose and glucose.

The The yy gene codes for permease, which increases gene codes for permease, which increases permeability of the cell to galactosides. permeability of the cell to galactosides.

The The aa gene encodes a transacetylase. gene encodes a transacetylase.

In addition to the structural genes the lac operon In addition to the structural genes the lac operon also hasalso has

regulatory genes:regulatory genes:

– Promoter: Binding site for RNA polymerasePromoter: Binding site for RNA polymerase

– Operator: Binding site of repressorOperator: Binding site of repressor

Control of lac operon Control of lac operon expressionexpression The control of the lac operon occurs by both The control of the lac operon occurs by both

positive and negative control mechanisms.positive and negative control mechanisms.

Negative control of the lac operonNegative control of the lac operon

What happens to lac operon when What happens to lac operon when glucose is present and lactose is absent?glucose is present and lactose is absent?

During normal growth on a glucose-based During normal growth on a glucose-based medium (lacking lactose), the medium (lacking lactose), the laclac repressor is repressor is bound to the operator region of the bound to the operator region of the laclac operon, preventing transcription. operon, preventing transcription.

What happens when glucose is absent What happens when glucose is absent and lactose is present?and lactose is present?

The few molecules of lac operon enzymes The few molecules of lac operon enzymes present will produce a few molecules of present will produce a few molecules of allolactose from lactoseallolactose from lactose..

Allolactose is the inducer of the Allolactose is the inducer of the laclac operon.operon.

The inducer binds to the repressor The inducer binds to the repressor causing a conformational shift that causes causing a conformational shift that causes the repressor to release the operator.the repressor to release the operator.

With the repressor removed, the With the repressor removed, the RNA polymerase can now bind RNA polymerase can now bind the promoter and transcribe the the promoter and transcribe the operon.operon.

Positive Control of the lac Positive Control of the lac operonoperon

What happens when both glucose and What happens when both glucose and lactose levels are high?lactose levels are high?

Since the inducer is present, the lac operon will Since the inducer is present, the lac operon will be transcribed.be transcribed.

However the rate of transcription is very slow However the rate of transcription is very slow (almost repressed) because glucose levels are (almost repressed) because glucose levels are high and therefore cAMP levels are low.high and therefore cAMP levels are low.

The repression of the The repression of the laclac operon under these operon under these conditions is termed catabolite repression and is conditions is termed catabolite repression and is as a result of the low levels of cAMP that results as a result of the low levels of cAMP that results from an adequate glucose supply. from an adequate glucose supply.

This repression is maintained until the glucose This repression is maintained until the glucose supply is exhausted. supply is exhausted.

What happens when glucose levels start What happens when glucose levels start dropping in the presence of lactose?dropping in the presence of lactose?

As the level of glucose in the medium falls, the As the level of glucose in the medium falls, the level of cAMP increases. level of cAMP increases.

Simultaneously the inducer (allolactose) is also Simultaneously the inducer (allolactose) is also binding to the binding to the laclac repressor (since lactose is repressor (since lactose is present). present).

The net result is an increase in The net result is an increase in transcription from the operon. transcription from the operon.

The ability of cAMP to activate The ability of cAMP to activate (increase) expression from the (increase) expression from the laclac operon results from an interaction of operon results from an interaction of cAMP with a protein termed cAMP with a protein termed CRPCRP (for (for cAMP receptor protein). cAMP receptor protein).

The protein is also called The protein is also called CAPCAP (for (for catabolite activator protein).catabolite activator protein).

The cAMP-CAP complex binds to a region of the The cAMP-CAP complex binds to a region of the laclac operon just upstream of the promoter operon just upstream of the promoter

The binding of the cAMP-CRP complex to the The binding of the cAMP-CRP complex to the laclac operon stimulates RNA polymerase activity 20-to-operon stimulates RNA polymerase activity 20-to-50-fold. 50-fold.

(Repression of the (Repression of the laclac operon is relieved in operon is relieved in the presence of glucose if excess cAMP is the presence of glucose if excess cAMP is added.) added.)

cAMP is therefore an activator of the lac operon.cAMP is therefore an activator of the lac operon.

This type of regulation by an activator is positive in This type of regulation by an activator is positive in contrast to the negative control exerted by contrast to the negative control exerted by repressors.repressors.

trp operontrp operon

The The trptrp operon encodes the genes for the synthesis of tryptophan. operon encodes the genes for the synthesis of tryptophan. As with all operons, the As with all operons, the trptrp operon consists of the promoter, operator operon consists of the promoter, operator

and the structural genes. and the structural genes.

It is also subject to negative control by a repressorIt is also subject to negative control by a repressor

In this system, unlike the In this system, unlike the laclac operon, the gene for the repressor is not operon, the gene for the repressor is not adjacent to the promoter, but rather is located in another part of the adjacent to the promoter, but rather is located in another part of the E. coli E. coli genome. genome.

Another difference is that the operator resides entirely within the Another difference is that the operator resides entirely within the promoter promoter

Unlike an inducible system, the repressible operon is usually turned Unlike an inducible system, the repressible operon is usually turned on.on.

Structure of the trp operonStructure of the trp operonThe operon consists of:The operon consists of:

The operon consists of 5 structural genes that The operon consists of 5 structural genes that code for the three enzymes required to convert code for the three enzymes required to convert chorismic acid into tryptophan’chorismic acid into tryptophan’

The operon also contains a gene coding for a short The operon also contains a gene coding for a short oligopeptide (oligopeptide (trpLtrpL) which functions in attenuation ) which functions in attenuation

OperatorOperator

promoterpromoter

Gene Gene Gene Function Gene Function

PP/O /O Promoter; operator sequence is found in the Promoter; operator sequence is found in the promoter promoter trp Ltrp L Leader sequence; containing attenuator (A) Leader sequence; containing attenuator (A) sequence the leader sequence the leader

trp Etrp E Gene for anthranilate synthetase subunit Gene for anthranilate synthetase subunit

trptrp DD Gene for anthranilate synthetase subunit Gene for anthranilate synthetase subunit

trptrp CC Gene for glycerolphosphate synthetase Gene for glycerolphosphate synthetase

trp Btrp B Gene for tryptophan synthetase subunit Gene for tryptophan synthetase subunit

trp Atrp A Gene for tryptophan synthetase subunit Gene for tryptophan synthetase subunit

Negative control of trp operonNegative control of trp operon

The affinity of the The affinity of the trptrp repressor for binding the repressor for binding the operator region is enhanced when it binds operator region is enhanced when it binds tryptophan, blocking further transcription of the tryptophan, blocking further transcription of the operon and, as a result, the synthesis of the three operon and, as a result, the synthesis of the three enzymes will decline.enzymes will decline.

hence tryptophan is a corepressor. hence tryptophan is a corepressor.

This means that when tryptophan is absent This means that when tryptophan is absent expression of the trp operon occursexpression of the trp operon occurs

the rate of expression of the the rate of expression of the trptrp operon is graded in operon is graded in response to the level of tryptophan in the cell. response to the level of tryptophan in the cell.

Attenuation of the Attenuation of the trptrp operon operon

Expression of trp operon is reduced by the Expression of trp operon is reduced by the addition of trytophan in trpR mutants.addition of trytophan in trpR mutants.

Further research established that this second Further research established that this second level of tryptophan control involved two level of tryptophan control involved two components:components:

1.1. tRNA, specifically tryptophanyl-tRNA, specifically tryptophanyl-tRNA, tRNATrp, i.e. tRNATrp charged tRNA, tRNATrp, i.e. tRNATrp charged with tryptophan.with tryptophan.

2.2. the the trpLtrpL gene   gene  

The attenuator region is composed of The attenuator region is composed of sequences found within the transcribed RNA of sequences found within the transcribed RNA of the operonthe operon

It is involved in controlling transcription from It is involved in controlling transcription from the operon after RNA polymerase has initiated the operon after RNA polymerase has initiated synthesis of the proteins. synthesis of the proteins.

The leader sequences are located prior to the The leader sequences are located prior to the start of the coding region for the first gene of start of the coding region for the first gene of the operon (the the operon (the trpEtrpE gene). gene).

The leader sequence (trp L) contains tandem The leader sequence (trp L) contains tandem tryptophan codons. tryptophan codons.

How does this affect transcription of the trp How does this affect transcription of the trp operon?operon?

It contains two consecutive It contains two consecutive trptrp codons and codons and therefore serves to measure the tryptophan therefore serves to measure the tryptophan supply in the cell. supply in the cell.

If the supply is good, then the tRNA will be If the supply is good, then the tRNA will be charged and the leader peptide will be translated charged and the leader peptide will be translated without problem. without problem.

If the supply is inadequate, then the tRNA will not If the supply is inadequate, then the tRNA will not be charged, and translation will stall at the be charged, and translation will stall at the trptrp codons. codons.

The trpL mRNA region can adopt a number of The trpL mRNA region can adopt a number of different conformations. It contains several self-different conformations. It contains several self-complementary regions which can form a variety complementary regions which can form a variety of stem-loop structures of stem-loop structures

Different stem-loops can form:Different stem-loops can form:

– Depending on the level of tryptophan in the Depending on the level of tryptophan in the cell and hence the level of charged trp-tRNAscell and hence the level of charged trp-tRNAs

– Depending on the position of ribosomes on the Depending on the position of ribosomes on the leader polypeptide and leader polypeptide and

– Depending on the rate at which they are Depending on the rate at which they are translated translated

trptrp Operon Transcription Under Operon Transcription Under High Levels of TryptophanHigh Levels of Tryptophan

Recall that transcription and translation can Recall that transcription and translation can occur simultaneously in bacteria. occur simultaneously in bacteria.

This means that the ribosome will attach to This means that the ribosome will attach to mRNA and is able to influence the formation of mRNA and is able to influence the formation of secondary structures by the mRNA.secondary structures by the mRNA.

In the case of the In the case of the trpLtrpL mRNA, when the cellular mRNA, when the cellular levels of tryptophan are high, the levels of the levels of tryptophan are high, the levels of the tryptophan tRNA are also high. tryptophan tRNA are also high.

Immediately after transcription, the ribosome Immediately after transcription, the ribosome follows right behind RNA polymerase until it is follows right behind RNA polymerase until it is halted by a stop codon. halted by a stop codon.

Translation is quick because of the high levels of Translation is quick because of the high levels of tryptophan tRNA. tryptophan tRNA.

This permits formation of the terminator stem-loop This permits formation of the terminator stem-loop which will cause RNA polymerase to dissociate which will cause RNA polymerase to dissociate (recall rho independent termination of transcription (recall rho independent termination of transcription in prokaryotes)in prokaryotes)

How is the terminator stem-loop How is the terminator stem-loop formed? formed?

Because of the quick translation of domain 1, domain Because of the quick translation of domain 1, domain 2 becomes associated with the ribosome complex. 2 becomes associated with the ribosome complex.

Then domain 3 binds with domain 4, and Then domain 3 binds with domain 4, and transcription is attenuated because of this stem loop transcription is attenuated because of this stem loop formation.formation.

The stem loop formed by binding of domains 3 and 4 The stem loop formed by binding of domains 3 and 4 is found near a region rich in uracil and acts as the is found near a region rich in uracil and acts as the transcriptional terminator loop (see transcription transcriptional terminator loop (see transcription notes from unit 3)notes from unit 3)

Consequently, RNA polymerase is dislodged from the Consequently, RNA polymerase is dislodged from the template. template.

trptrp Operon Transcription Under Operon Transcription Under Low Levels of TryptophanLow Levels of Tryptophan

Under low cellular levels of tryptophan, the translation of the Under low cellular levels of tryptophan, the translation of the short peptide on domain 1 is slow. short peptide on domain 1 is slow.

As a result domain 2 does not become associated with the As a result domain 2 does not become associated with the ribosome. ribosome.

Rather domain 2 of the leader mRNA associates with domain 3 Rather domain 2 of the leader mRNA associates with domain 3 of the leader mRNA. of the leader mRNA.

This step loop structure is the anti-terminator. Its formation This step loop structure is the anti-terminator. Its formation prevents formation of the terminatorprevents formation of the terminator

This structure permits the continued transcription of This structure permits the continued transcription of the operon. Then the the operon. Then the trpE-AtrpE-A genes are translated, and genes are translated, and the biosynthesis of tryptophan occurs the biosynthesis of tryptophan occurs

Domain 4 is called the Domain 4 is called the attenuatorattenuator because its because its presence is required to reduce (attenuate) mRNA presence is required to reduce (attenuate) mRNA transcription in the presence of high levels of transcription in the presence of high levels of tryptophan. tryptophan.

Domain 1 is also an important component of the Domain 1 is also an important component of the attenuation process. attenuation process.

The section of the leader sequence encodes a 14 The section of the leader sequence encodes a 14 amino acid peptide that has two tryptophan residues. amino acid peptide that has two tryptophan residues.

ReferencesReferences

http://www.biocourse.com/ui/swf/http://www.biocourse.com/ui/swf/iLabs/lac_operon.swfiLabs/lac_operon.swf

http://faculty.plattsburgh.edu/donhttp://faculty.plattsburgh.edu/donald.slish/Att-Trp.htmlald.slish/Att-Trp.html

http://highered.mcgraw-hill.com/sihttp://highered.mcgraw-hill.com/sites/dl/free/0072835125/126997/ates/dl/free/0072835125/126997/animation28.htmlnimation28.html