Regulation of Gene Regulation in Bacteria Chapter 15

gene regulation

The transcription of a gene is tightly controlled in living organisms. This includes the abundance (how much) of the transcript and the timing (when) of the transcription

Gene regulation network is the

"electronic board" behind the cellular function

Prokaryotes synthesize only the proteins that are needed

•Gene expression is tightly regulated-genes whose products are not needed are not expressed
• Different genes are expressed under different environmental conditions
Efficiency! Efficiency! Efficiency!

Key DNA sequences in Prokaryotic transcription Regulation

1. Promoter
2. mRNA-coding sequence
3. Terminator

Promoter.

Upstream of RNA coding sequence. Sites where RNA Pol interacts with DNA to begin transciption

mRNA-coding Sequence

DNA strand that is transcribed by RNA Pol into a RNA transcript

Terminator

Downstream of the RNA coding sequence that Specifies where transcription will stop

In Prokaryotes, some genes are constitutive

They are always actively transcribed because their products are essential to the normal cellular function regardless of the conditions.

Inducible Genes

Genes that are turned on in response to an extracellular signal

- Inducer is the substance that is responsible for inducing gene expression

Repressible Genes

Genes that are turned off in response to an extracellular signal

- Suppressor is often the end product of a biosynthetic pathway

Negative Regulation

Genes are on until turned off by a signal, or regulatory molecule

Positive Regulation

Genes are off until turned on by a signal, or regulatory molecule

Gene transcription is regulated in units of

operon

Operon

Clustering of genes that are transcribed together and the adjacent regulatory elements

Promoter

regulatory DNA sequences required for gene transcription

operator

a regulator DNA sequence that control the gene expression, usually by interacting with a regulatory protein

Structural genes

function in the same pathway and are adjacent to each other on the chromosome. These genes are often transcribed together in a single mRNA molecule called Polycistronic mRNA

Inducible genes are expressed

only when induced

Lactose Metabolism in E. coli.

Level of expression is directly proportional to Lactose concentration in media

-Not produced by the cell in absence of lactose
-Rapidly induced (about 1000-fold) in the presence of lactose
-Expression is repressed when lactose is used up
-Induction of all 3 enzymes is coordinate-part of polycistronic operon

Lactose is broken down into glucose and galactose by

beta-galactosidase

Galactose is subsequently converted to

glucose to be utilized by the cell

The structural Genes of the Lac Operon
Expression of the structural genes

of the lac operon genes are transcribed into a single polycistronic mRNA, which is translated simultaneously into the three enzymes encoded by the operon

The structural Genes of the Lac Operon
Mutations in the structural genes

Produce a lac- phenotype cells are not able to utilize lactose as a carbon source

The lac Operon Model

I P O Z Y A

(I) repressor gene
(P) promoter
(O) operator gene
(L) Leader
(Z)
(Y) structural genes
(A)

When Lactose is absent

Repressor-Binds to DNA in the operator and inhibits the ability of RNA pol to bind to the promoter and activate transcription
-No transcription and No expression of structural genes

Operator - cis-acting element
-Must be adjacent to (upstream) of the operon to function

Repressor - trans-acting factor
-Diffusible factor (protein) - binds to the operator

When lactose is present

lac operon switched on to allow transcription

• Lactose binds to the repressor and alters the conformation of the repressor
• Allosteric conformation change - binding of allolactose to repressor
• Repressor can no longer bind to DNA
• RNA Pol is now able to transcribe the structural genes
• Enzymes needed for lactose metabolism are produced

Absence of lactose

-LacI binds to the lacO operator sequence LacI blocks acc
-ess of RNA Pol to the operon

Presence of lactose

-Lactose binds to LacI repressor and change its conformation through Allosteric interaction.
-LacI loses its affinity for the operator and falls off
-In the absence of the repressor, RNA Pol can bind to the promoter and transcribe the operon

Mutations of the repressor
When a cell bears the I- mutation.

-Mutation in the gene encoding the LacI repressor protein
-No repressor protein is produced
-results in constitutive expression of the lac operon
-lac operon expressed even in the absence of lactose (and presence of glucose)

Lac I- Mutations: Loss of function - constitutive expression

Mutations of the repressor
When a cell bearing the IS mutation

-Mutant repressor molecules are produced that can't bind the inducer.
-So repressor is always bound to the operator sequence
-Structural genes are permanently repressed

Mutations of the operator
When a cell bears the lac O c mutation

-Mutation in the Operator region
-Alteration of the nucleotide sequence within this region results in the inability of the normal, wt repressor protein to recognize and bind to the operator element
-Result is the same as with the lacI- mutation: Constitutive expression of the lac operon structural genes

LacI - must bind to a particular DNA sequence

Mutations that alter expression of the lac Operon
Mutation in Repressor Gene:

- lacI- - Constitutive expression of lac operon, Can't bind operator
- lacIS - Super-repressor - Mutant repressor molecules that cannot bind the inducer, repressor is always bound to the operator. No expression of the lac operon

Mutations that alter expression of the lac Operon
Mutation in Operator Region

LacO C - Constitutive expression of lac operon, nucleotide sequence of operator DNA is altered

Mutations that alter expression of the lac Operon
Mutations in Structural Genes

lacZ-; lacY-; lacA- - Cells with mutations in any of these genes are unable to breakdown lactose

What happens to expression of the lac operon when cells are grown in the presence of both glucose and lactose?

Glucose is the preferred carbon source.

Expression of Lac operon is repressed by Catabolite Repression

- Mediated by Catabolite-Activating Protein (CAP), helps activate expression of lac operon, but is able to inhibit expression when glucose is present- catabolite repression
- In presence of glucose, CAP activity is inhibited resulting in inefficient binding of RNA Pol to the promoter

Positive Control of the lac Operon-Catabolite Repression

Role of Glucose in Catabolite Repression
• Glucose inhibits the activity of the enzyme Adenyl cyclase which Converts ATP into cAMP
• In absence of cAMP, CAP cannot form the CAP-cAMP complex needed for activation of the lac operon

Catabolite Repression of the lac Operon

-lac operon is repressed when glucose and lactose are present
-when glucose is used up, lactose inducible enzymes are then induced
-transcription of lac operon requires both that lactose be present and that glucose not be present

slide 26 chart

Predict the level of genetic activity of the lac operon as well as the status of the lac repressor and the CAP protein under the cellular conditions listed below

Prokaryotic gene regulation II: Trp Operon
The advantage of such regulation

when sufficient amounts of a particular amino acid are present in the growth media, the genes encoding the enzymes for that biosynthetic pathway are turned off: Bacteria can synthesize all necessary amino acids if needed (not provided in growth media)

Trp Operon - Repressible operon

The gene transcription is repressed in the presence of tryptophan

Trp Operon - Repressible operon
Expression is repressed by

the presence of a chemical (amino acid), in contrasts with lac operon which is turned on by the presence of a chemical (lactose)

Components of the trp Operon

1. Regulatory region
2. Trp Repressor
3. Structural genes

Components of the trp Operon
Regulatory region

Promoter and operator regions are upstream from the structural genes. Additionally, there are the leader and attenuator sequences

Components of the trp Operon
Trp Repressor

Trp repressor binds to operator in presence of tryptophan. They represses operon expression, as Trp participates in repression, called corepressor

Components of the trp Operon
Structural genes

Five, Transcribed as a polycistronic mRNA

In the absence of tryptophan

-An inactive repressor is made that cannot bind to the operator (O)
-Allows transcription to proceed

In the presence of tryptophan

-It binds to the repressor, causing an allosteric transition to occur.
-This complex binds to the operator region, leading to repression of the operon

Mutations that alter expression of the trp operon
trpR -

no repressor produced
-expression of the trp operon is constitutive
-Expression of wt trpR gene in trpR- cells restore

Mutations that alter expression of the trp operon
trpO C

Operator region mutations
- Constitutive expression of the trp operon
-Addition of a wild type operator as a trans
-acting element does not restore regulation of the trp operon repression

Leader mRNA

-can be translated into a short polypeptide -contains two trp codon just before a stop codon -Four regions in the leader mRNA fold into alternative secondary structures

Transcription of Trp operon

Trp starvation:

-Amount of trp-tRNA drops and the ribosome pauses at the two trp codons in the leader mRNA
-translation of the leader peptide cannot be completed

Transcription of Trp operon

Ribosome pausing - covers region 1

-1 and 2 pairing cannot occur
-Region 2 pairs with region 3
-Prevents region 3 and 4 pairing
-2-3 pairing signals antitermination
-RNA Pol continues past the attenuator and transcribes the structural genes

Termination of trp operon transcription

Enough Trp is present

-the ribosome translates the leader peptide to its stop codon
-The ribosome covers parts of region 2, cannot pair with region 3
-Allows Region 3 to pair with region 4

-Termination signal
-The 3-4 structure is the attenuator
-causes RNA Pol to terminate transcription

Regulation by attenuation

-Transcription and Translation are coupled in Prokaryotes: Leader peptide synthesis occurs just behind the RNA Pol, Allows the process of attenuation to occur
- Key Signal is the cellular concentration of tRNAtrp.

Regulation by Attenuation

High trp concentrations
- leader sequence is translated, but full length mRNA is not transcribed
- transcription is terminated prematurely

Low trp concentration
- repressor is not bound to promoter
-transcription proceeds through the leader and transcribes the full-length mRNA, leader sequence is not translated
-No premature transcription termination

Riboswitches

Folded RNAs that act as switches regulating protein synthesis in response to environmental conditions

All riboswitches possess metabolite-sensing RNA sequence

Allows transcription of RNA to proceed or not

Two important domains within riboswitch

- Aptamer: binds to ligand
- Expression platform: capable of forming terminator structure

Riboswitch allows metabolite to

regulate mRNA transcription

Binding between ligand and short RNA sequence leads to

- Alternative forms of mRNA secondary structure
- Bind with small ligands; cause conformational change and induce second RNA domain
- Create antiterminator or terminator structure

CRISPR/Cas9—

Molecular mechanism by which bacteria respond to specific bacteriophage attack.
- Leads to destruction of invading phage DNA

a revolutionary gene editing technique derived from the immune system of simple prokaryotes

Genomic locus in bacteria that contains

- Clustered Regulatory Interspaced Short Palindromic Repeats
- First identified in E. coli genome.
• Repeated DNA sequences with nonrepetitive spacer sequences between them.
- CRISPR loci now identified in ~50% of bacteria and ~90% of archaea.

Mechanism of CRISPR-Cas

Step 1: Spacer acquisition
Step 2: CRISPR-derived RNAs (crRNAs)
Step 3: Target interference

Step 1: Spacer acquisition

- Invading phage DNA cleaved into small fragments.
- Directly inserted into CRISPR locus—become new spacers.
- Cas1 nuclease and Cas2 protein required for spacer acquisition.
- New spacers inserted closer to leader sequence of CRISPR locus

Step 2: CRISPR-derived RNAs (crRNAs)

- CRISPR loci transcribed
- Long transcripts processed into crRNAs.
- Each containing single spacer and repeat sequence—crRNA biogenesis.

Step 3: Target interference

- Mature crRNAs associate with Cas nucleases.
- Recruited to complementary sequences in invading phage DNA.
- Cas nucleases cleave viral DNA — infection neutralized.