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Regulation of gene Regulation of gene expression in expression in

Prokaryotic cellsProkaryotic cells

Ratchada Cressey, Ph.DRatchada Cressey, Ph.DAssistant ProfessorAssistant Professor

Clinical ChemistryClinical Chemistry

Associated Medical ScienceAssociated Medical Science

Chiang Mai UniversityChiang Mai University

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Comparison of Prokaryotic and Comparison of Prokaryotic and Eukaryotic cell structureEukaryotic cell structure

Prokaryotic Cell ( Prokaryotic Cell ( Bacillus megaterium Bacillus megaterium))

-Eukaryotic Cell (L Cell) -Eukaryotic Cell (L Cell)

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Comparison of Prokaryotic and Comparison of Prokaryotic and Eukaryotic gene structureEukaryotic gene structure

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Logic of gene expressionLogic of gene expression

EukaryotesEukaryotes• Eukaryotes are (mostly) metazoan

– Colonies of specialized cells– Almost all cells die at the end of generation– Only gamates survive- they do not respond

to environmental stimuli

• Most cells provide a specialized function• Few cells are involved in responses to

environmental stimuli

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Logic of gene expressionLogic of gene expression

ProkaryotesProkaryotes• Cells respond to fast environmental

changes• Must compete for carbon sources• Changes in gene may ‘persist’ for

several generations• Gene expression is capable of

responding to signals not seen in many generations

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Gene Regulation in BacteriaGene Regulation in Bacteria

• Bacteria adapt to changes in their surroundings by using regulatory proteins to turn groups of genes on and off in

response to various environmental signals.

• The DNA of Escherichia coli is sufficient to encode about4

000 proteins, but only a fraction of these are made at a ny one time. E. coli regulates the expression of many of its gene

s according to the food sources that are available to it.

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Gene Structure of bacteria

Transcription start site

Transcription

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consensus

TATA (Pribnow) box

E. coli Promoters

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Gene expression is regulated in many Gene expression is regulated in many different ways in prokaryotesdifferent ways in prokaryotes

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Method for studying of DNA-Method for studying of DNA-protein interactionprotein interaction

• EMSA (Electrophoresis Mobility Shift Assay) or gel shift assay

• DNA footprinting– DNAse I footprinting

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Gel shift assayGel shift assay

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1. Prepare end-labeled DNA.

2. Bind protein.3. Do a mild digestion with

DNAse I (Dnase I randomly cleaves DS DNA on each strand)

4. Separate DNA fragments on denaturing acrylamide gels (sequencing gels)

5. Expose gel to X-Ray film.

2. DNAse I Footprinting

Fig. 5.37a

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Sample of a DNase I footprinting gel (for a DNA-binding protein).

Footprint

Samples in lanes 2-4 had increasing amounts of the DNA-binding protein (lambda protein cII); lane 1 had none.

Fig. 5.37b

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Differences between prokaryotes Differences between prokaryotes

and eukaryotes:and eukaryotes:

• Prokaryote gene expression typically is regulated by an operon, the collection of controlling sites adjacent to protein-coding sequence.

• Eukaryotic genes also are regulated in units of protein-coding sequences and adjacent controlling sites, but operons are not known to occur.

• Eukaryotic gene regulation also is more complex because eukaryotes possess a nucleus.(transcription and translation are not coupled).

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What is An Operon?What is An Operon?• As we have learned, Operons aid or

repress the transcription proteins.• Operons are made up of the Operator, and

the genes it controls.• Operons are synthesized into a single

molecule of mRNA that holds the information for the Prokaryote to transcribe.

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How do Operons Regulate?How do Operons Regulate?• Negative

Control – mRNA synthesis proceeds more rapidly in the absence of the active controlling factor.

• Positive Control – Operons only function in the presence of a controlling factor.

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Induction and Repression of Induction and Repression of bacterial enzymebacterial enzyme

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A Little History First….A Little History First….• In 1961, Jacob and Monod found a protein,

a repressor, that could control the production of -galactosidase. They believed that this protein worked when bonded to an operator. They named this complex the Lac Operon, and won the Nobel Prize in 1964.

                               

François Jacob

                               

Jacques Monod

                     

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1. Inducible System1. Inducible System• Inducers – raise the levels of inducible

enzymes.• Repressor Proteins – repress mRNA

synthesis, this is the active control factor.• Inducers – Bind with the repressor, making

it inactive and allowing transcription to take place, to create, the inducible enzyme.

No Inducers = No Enzymes = No Metabolism

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The Lactose OperonThe Lactose Operon• Negative Control System in E. Coli• Gives a good example of a Inducible

System.

        

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Regulation of the Lac operonRegulation of the Lac operon

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The Lac Inducible System The Lac Inducible System Negative controlNegative control

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How does this inhibit How does this inhibit transcription?transcription?

• The Promoter, where the RNA polymerase binds is located next to the Operator, when a repressor binds to it, it bends the DNA so the RNA polymerase will not bind or can not begin to transcribe.

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The binding of repressor to the The binding of repressor to the Lac operonLac operon

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The Lac Inducible SystemThe Lac Inducible SystemRepressor is Turned OffRepressor is Turned Off

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• Lactose itself was not the induce Lactose itself was not the inducerr but several galactosides which are but several galactosides which are not metabolized would work. not metabolized would work. •allolactoseallolactose was found to be the physiological inducer. It is a secondary was found to be the physiological inducer. It is a secondary - metabolite of lactose as a byproduct of basal ß galactosidase activity - metabolite of lactose as a byproduct of basal ß galactosidase activity

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IPTGIPTG (isopropylthiogalactoside) is a good , (isopropylthiogalactoside) is a good , non-metabolized inducer. non-metabolized inducer.

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What about Positive What about Positive Control?Control?

• Operons function when the controlling factor is present.

• The Lac Operon is also good example of Positive control.

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cAMPAMP

AC

Adenylate cyclase and CAP mediate glucose repression of Lac

Adenylate cyclase (AC) is an enzyme that synthesizes cyclic AMP (cAMP) from ATP

High glucose adenylate cyclase is inhibited (indirectly, via a catabolic product) Therefore cAMP levels are LOW

Absence of glucose adenylate cyclase is NOT repressed. Therefore cAMP levels are HIGH

cAMP forms a complex with the CAP protein, which allows it to then bind to the CAP site upstream of the Lac operon. Binding of the CAP protein is required to allow RNA polymerase to bind to the lac promoter and turn on transcription. In the absence of CAP binding, there is no (or very little) transcription of the lactose operon, even in the presence of lactose.

glucose

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cAMP Regulartory Protein (CRP)cAMP Regulartory Protein (CRP)

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cAMP Regulartory Protein (CRP)cAMP Regulartory Protein (CRP)

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Interaction of cAMP, CAP, and the Lac RepressorInteraction of cAMP, CAP, and the Lac Repressor

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cAMP binding causes cAMP binding causes conformational change of CRPconformational change of CRP

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CAP mediates glucose repression of Lac

Promotes transcription

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Lactose Glucose

- +

- -

+ -

+ +

LacI

CAP-cAMP

Four States of the Lac Operon

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2. Repressible System2. Repressible System• Aporepressor – the inactive form of a

repressor.• Corepressors – bind to the aporepressor,

and make it an active repressor.• Repressible Systems – enzymes are

reduced by the presence of the end product.

• Good example is the Tryptophan operon.

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Tryptophan operon: Tryptophan operon: Regulation by Regulation by repression repression and and attenuationattenuation

• Genes for tryptophan synthesis• Repressed by end-product of pathway,

Tryptophan.• Repression requires Operator sequence,

Aporepressor (trpR gene product) & Co-repressor (Tryptophan).

• Also controlled by attenuation in the “Leader” peptide region of the transcript.

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Tryptophan operonTryptophan operon

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Tryptophan OperonTryptophan Operon

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Attenuation provides secondary control Attenuation provides secondary control mechanism in the Trp operon mechanism in the Trp operon

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Attenuation provides secondary control Attenuation provides secondary control mechanism in the Trp operon mechanism in the Trp operon

• Attenuation – the premature termination of transcription.

• Leader Region – lies between the Operator and the 1st structural gene. It contains four segments we will call 1, 2, 3, 4.

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Tryp operon: Repressible controlTryp operon: Repressible control

• Segment 1 contains 2 trp codons.

• If tryptophan levels are low, translation in segment 1 are slow therefore segment 2 is not bound by ribosomes, and is free to hairpin with segment 3, and transcription occurs.

• If trytophan levels are high, the tryptophanyl-tRNA is available for proteins synthesis, ribosomes will to bind with segment 2, therefore 3 and 4 hairpin to create a termination site.

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The trp Leader peptide has two key tryptophan codons.

The ribosome stalls at the trp codons when [Tryptophan] is too low.The stalled ribosome prevents a downstream transcription terminator (IR + U-rich sequence) from forming.

Fig. 7.35

49Fig. 7.36

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Mechanism of AttenuationMechanism of Attenuation

• rU-dA base-pairs are exceptionally weak, they have melting temperature 20C lower than rU-rA or dT-rA

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Attenuation vs. No AttenuationAttenuation vs. No Attenuation

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The Arabinose Operon

• Ara operon: 3 structural genes required to utilize the sugar arabinose (araB, araA and araD).

• Regulator protein araC

• AraC turns on transcription of the ara operon by binding to the araI initiatior site only when it is bound to arabinose

• In the absence of arabinose, araC protein undegoes a conformation change Now binds to BOTH araI and araO, which forms a loop that inhibits transcription.

• The Ara operon is also subject to catabolite repression.

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Arabinose OperonArabinose Operon

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The Arabinose Operon

This loop prevents RNA transcription (NOT true for all loops)

No Arabinose present, operon OFF

Arabinose present, Glucose absent, operon ON

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The Arabinose OperonThe Arabinose Operon

RNA polymerase

Arabinose

AraC

CAP-cAMP

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Autoregulation of araCAutoregulation of araC

• As the level of AraC (green) rises, it binds to araO1 and prevent transcription leftward from Pc through the araC gene, thus preventing anaccumulation of too much repressor

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Promoters and Sigma Factors

• Part of the RNA polymerase enzyme that recogniz es the promoter is called the sigma factor. After tr

anscription begins, this unit dissociates from the enzyme

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Different sigma factors recognize Different sigma factors recognize different promotersdifferent promoters

• Different sigma factors recognize different promoters and thus, the availability of sigma factors can regulate the transcription of genes associated with these

promoters.

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Example of Translational Control in Prokaryotes: Antisense RNA

• Normally, mRNA is synthesized off of the template (antisense ) strand of DNA. Antisense RNA is synthesized from the nonco

ding (sense) strand of DNA. The two mRNA molecules bond to gether, inactivating the mRNA

• This mechanism appears to be universal among bacteria. It h as not been shown to be a normal means in eukaryotes

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Suggested reading:Suggested reading:

• Robert F. Weaver, Molecular biology, second edition, 2002