Chapter 17Regulation of Gene Expression in Bacteria and Bacteriophages

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Chapter 19: Regulation of Gene Expression in Bacteria and Bacteriophages

Through evolutionary processes, organisms have developed ways to compensate for environmental changes.

Alter gene activity to optimize growth and reproduction in a given environment.

Two Types of Genes

1) Regulated Genes – activity is controlled in response to the needs of a cell or organism.

2) Constitutive genes - (housekeeping genes) always active (e.g. protein synthesis and Glucose metabolism)

Basic Mechanism of Gene Regulations in Bacteria

Bacteria have developed ways to turn off genes whose products are not needed and for turning on genes whose products are needed in each environment.

The turning of genes off or on requires interaction between regulatory proteins and DNA sequences.

Inducible Gene Expression

When a gene is turned on by the addition of a substance, it is called inducible gene.

The regulator substance is called an inducer, which are members of a class of small molecules = effectors

Controlling site is near protein coding sequence.The addition of inducer leads to induction.

Induction of genes required for lactose utilization in E. coli

E. coli grow in simple media containing salts, nitrogen sources, and glucose.

If lactose (or other sugar) is added instead of glucose,

a number of enzymes are rapidly synthesized.

When Lactose is only sugar, three enzymes are synthesized1) B-galactosidase2) Lactose permease3) Trans acetylase (function poorly understood)

*Inducer of Lactose operon

All 3 genes are clustered on the genome and are transcribed onto a single mRNA called a polygenic mRNA or a polycistronic mRNA

Nonsense mutants (chain terminating mutant) were used to determine that all 3 genes were on the same mRNA.

Chain terminating mutation

Jacob & Monod’s Operon Model for the regulation of the lac genes

• Operon is a cluster of genes, the expression of which are regulated together by operator regulator protein interactions, plus the operator region itself and the promoter.

In E. coli, the lac operon is under negative, positive and inducible control

Lac I+ gene encodes lac repressor protein made constitutively, which will bind operator region of the lac operon.

Few repressors are present in cell since promoter is relatively weak.

Absence of lactose

Lac operon is under negative control:

There is a low level of lac gene expression because the repressor binds and unbinds allowing for low amounts of protein such as B-galactosidose and permease to be generated

Presence of lactose

Inducible Control

If in the presence of lactose, the above B-galactosidose produces inducer molecules, allolactose, which is the inducer.

Experiments by Jacob and MonodPartial diploid that has F‘ plasmid

Without inducer

With inducer

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Mutation in Lac I gene, which generates a mutant repressor that cannot bind to the operator

Withoutinducer

Mutation in Lac I gene, which generates a mutant repressor that cannot bind to the operator

With inducer

Lac Operon experiments

Dominant EffectMutation in Lac I that cannot bind Inducer but can bindthe operator

Lac Repressor model for tetramer protein structure

Has four polypeptides and each polypeptide is fromrepressor gene.

**This data convinced many scientist (at the time) that all genes were under negative control due to the binding of a repressor. **

Positive control of the lac operon. Turns on expression of the lac operon.

Ensures that lac operon stays on when lactose is the sole carbon source, but not in the presence of glucose.

Glucose is used preferentially over lactose.

Positive control of the lac operon.

In presence of glucose, concentration of positive regulator (CAP-cAMP complex) that binds the lac operon for increased gene activity is reduced.

Because the presence of glucose reduces the amount of cAMP in cell.

Glucose inactivates adenylate cyclase

Glucose removes remaining cAMP by Activating Phosphodiesterase

Shine-Dalgarno sequence

GUG start site

Basepair sequence of the lac I gene promoter

Basepair sequence of the controlling sites, promoter and operator, for the lac operon.

Tryptophan OperonAll necessary amino acids may not be present in a growth medium. If a specific amino acid is missing, the bacteria has certain operons that enable the bacterial cell to manufacture that amino acid.

For example the Tryptophan Operon

Has five structural genes

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Two mechanisms of regulations for tryptophan operon

# 1 Repressor/operator interaction with tryptophan as the effectormolecule: Tryptophan binds the aporepressor (trpR) and then binds operator to turn off the gene

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Two mechanisms of regulations for tryptophan operon

# 2 Attenuation Control Regulatory Leader region Determines if initiated transcripts include other structural genes or not.

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The biosynthetic pathway is catalyzed by a specific enzyme (at each step) which is coded by a specific gene or genes. Presence of tryptophan in medium keeps operon turned off

Operon called repressible operon (# 1). No transcripts in presenceof tryptophan.

# 2 Attenuation Control

Absence of tryptophan or in the presence of low amounts of tryptophan:

Under severe tryptophan starvation all long transcriptsgene activity at maximum

Under less severe situation gene Long and short transcriptsExpression at less than maximum

Greater the amount of tryptophan the greater the number of short transcriptsAttenuation controls = > terminates transcription producing short transcripts

mRNA

RNA polymerase response to these mRNA secondary structures

Transcription and translation are tightly coupled in prokaryotesAttenuation occurs at the mRNA level and can reduce transcription of trp-operon 8-or-10 fold.

*Last long enough for Ribosome to load onto mRNA.

Position of ribosome on leader transcript determines if transcription is terminated or not.

If starved for trytophan = lack trp-tRNA

If no trp-tRNA, ribosome stalls at trp codons. With Ribosome on region one, the 1-2 loop can’t form.

So, the 2-3 loop for antitermination forms. Thus region 3 can’t pair with region 4 and the RNA polymerase can now continues.

++ trptophan

Starved for tryptophan

Termination signalFor RNA polymerase,Which stops transcription

Transcription continues

Attenuated-controlled bacterial operons Regulation of other amino acid biosynthesis operons

Leader Peptides of other attenuated-controlled Bacterial operons

Isoleucine and leucine

The ara Operon of E. coli: Positive and Negative Control

At the same time that Jacob & Monod were doing their work,Englesberg, et. al. were studyingThe regulation of the arabinose(ara) operon of E. coli.

They found that instead of beingregulated with a negative control mechanism as seen in the lac operon, the ara operon was primarily under Positive control. Although their conclusion were not widely accepted,biochemical and molecular test proved that they were correct.

In the lac operon, allolactose would bind the repressor to remove it from the operator so that thePolymerase could bind the operator and start transcription.

In the ara operon, two molecules of AraC protein bind and act as a bridge from the operator (araO2 )And to the promoter region ara I1 which creates a loop that prevents the binding of CAP-cAMP. With the addition of arabionose, the arabionose bound AraC protein is allosterically modified to bind toara I2, which allows CAP-cAMP to bind the CAP site and positive regulate gene expression occurs.

However, for the ara operon to function, glucose can not bePresent. If present, it will eliminate cAMP and focus on theUtilization of glucose

**Positive regulation of activators is now known to occur in aVariety of prokaryotic systems and in all eukaryotes.

Regulation of gene expression in the lytic cycle and lysogeny in baceriophage lambda (λ)

Excellent model for developmental switches in eukaryotic systemsAfter λ infection of bacteria, a choice is made between lytic and lysogenic pathways

Bacteriophage Gene regulation

1) Linear chromosome is circularizedin host

2) Transcription begins at PL & PR

PL promoter for left early operon

PR promoter for right early operon

These promoters are on different DNAStrands.

Depends on a genetic switch, which involves competition between the products of the CI gene (the repressor) and the Cro gene (the Cro protein)

regulator of CI gene.

Left Right

Important info

cI geneCro geneN gene

N = protein is the antiterminatorthat allows RNA transcriptionpast transcription terminator signals.

Lysogeny Lytic cII protein stimulates synthesis of cI repressor which competes with Cro protein.

Integration of λ

Decision on which pathway is taken is determined by the

amount of λ repressor or Cro protein that is bound

to PR or OR region.

cI protein

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