Prokaryote Expression

Regulation of gene expression in Prokaryotic cells Ratchada Cressey, Ph.D Assistant Professor Clinical Chemistry Associated Medical Science Chiang Mai University

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Comparison of Prokaryotic and Eukaryotic cell structure

Prokaryotic Cell (Bacillus megaterium)

Eukaryotic Cell (L-Cell)

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Comparison of Prokaryotic and Eukaryotic gene structure

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Logic of gene expression

Eukaryotes • 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 4

Logic of gene expression

Prokaryotes • 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 5

Gene Regulation in Bacteria • Bacteria adapt to changes in their surroundings by using regulatory proteins to turn groups of genes on a nd off in response to various environmental signals.

• The DNA of Escherichia coli is sufficient to encode about

4000

proteins, but only a fraction of these are made at a ny one time. E. coli regulates the expression of many of its g enes according to the food sources that are available to it. 6

Gene Structure of bacteria

Transcription Transcription start site

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E. coli Promoters

consensus TATA (Pribnow) box 8

Gene expression is regulated in many different ways in prokaryotes

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Method for studying of DNAprotein interaction • EMSA (Electrophoresis Mobility Shift Assay) or gel shift assay • DNA footprinting – DNAse I footprinting

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Gel shift assay

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2. DNAse I Footprinting 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. Fig. 5.37a

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

Fig. 5.37b

Footprint

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

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Differences between prokaryotes 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). 15

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. 16

How do Operons Regulate? • Positive Control –

Operons only function in the presence of a controlling factor.

• Negative Control – mRNA

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

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Induction and Repression of bacterial enzyme

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A Little History First…. • In 1961, Jacob and Monod found a protein, a repressor, that could control the production of β -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 19

1. 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 20

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

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Regulation of the Lac operon

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The Lac Inducible System Negative control

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How does this inhibit 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 Lac operon

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The Lac Inducible System Repressor is Turned Off

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•Lactose itself was not the inducer but several galactosides which are not metabolized would work. •allolactose was found to be the physiological inducer. It is a secondary metabolite of lactose as a byproduct of basal ß-galactosidase activity 28

IPTG (isopropylthiogalactoside) is a good , 29 non-metabolized inducer.

What about Positive Control? • Operons function when the controlling factor is present. • The Lac Operon is also good example of Positive control.

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Adenylate cyclase and CAP mediate glucose repression of Lac Adenylate cyclase (AC) is an enzyme that synthesizes cyclic AMP (cAMP) from ATP

AC

AMP

glucose

cAMP

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 32 of lactose.

cAMP Regulartory Protein (CRP)

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cAMP Regulartory Protein (CRP)

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Interaction of cAMP, CAP, and the Lac Repressor

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cAMP binding causes conformational change of CRP

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CAP mediates glucose repression of Lac

Promotes transcription

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Four States of the Lac Operon LacI Lactose

Glucose

-

+

-

CAP-cAMP

+

-

+

+ 39

2. 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. 40

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Tryptophan operon:

Regulation by repression and attenuation • Genes for tryptophan synthesis • Repressed by end-product of pathway, Tryptophan. • Repression requires Operator sequence, Aporepressor (trpR gene product) & Corepressor (Tryptophan). • Also controlled by attenuation in the “Leader” peptide region of the transcript. 42

Tryptophan operon

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Tryptophan Operon

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Attenuation provides secondary control mechanism in the Trp operon

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Attenuation provides secondary control 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 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 tryptophanyltRNA 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. 48 Fig. 7.35

Fig. 7.36

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

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Attenuation 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 Operon

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The Arabinose Operon Arabinose present, Glucose absent, operon ON

No Arabinose present, operon OFF

This loop prevents RNA transcription (NOT true for all loops) 55

The Arabinose Operon

RNA polymerase

AraC

CAP-cAMP

Arabinose

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Autoregulation 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 an accumulation of too much repressor 57

Promoters and Sigma Factors

• Part of the RNA polymerase enzyme that recognizes the promoter is called the sigma factor . After transcription begins, this unit dissociates fr om the enzyme 58

Different sigma factors recognize different 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 fro m the noncoding (sense) strand of DNA. The two mRNA mol ecules bond together, inactivating the mRNA • This mechanism appears to be universal among bacteria. It has not been shown to be a normal means in eukaryotes 61

Suggested reading: • Robert F. Weaver, Molecular biology, second edition, 2002

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