Regulation of Gene Expression in Bacteria OPERONS Through

Regulation of Gene Expression in Bacteria OPERONS

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 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 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. *Inducer of Lactose operon When Lactose is only sugar, three enzymes are synthesized 1) B-galactosidase 2) Lactose permease 3) Trans acetylase (function poorly understood)

All 3 genes are clustered on the genome and are transcribed onto a single m. RNA called a polygenic m. RNA or a polycistronic m. RNA Chain terminating mutation Nonsense mutants (chain terminating mutant) were used to determine that all 3 genes were on the same m. RNA.

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 Monod Partial diploid that has F‘ plasmid Without inducer With inducer

Mutation in Lac I gene, which generates a mutant repressor that cannot bind to the operator Without inducer Power. Point® Layered Art

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

Lac Operon experiments Dominant Effect Mutation in Lac I that cannot bind Inducer but can bind the operator

Lac Repressor model for tetramer protein structure Has four polypeptides and each polypeptide is from repressor 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. Glucose inactivates adenylate cyclase Glucose removes remaining c. AMP by Activating Phosphodiesterase In presence of glucose, concentration of positive regulator (CAP-c. AMP complex) that binds the lac operon for increased gene activity is reduced. Because the presence of glucose reduces the amount of c. AMP in cell.

Basepair sequence of the lac I gene promoter Shine-Dalgarno sequence GUG start site Basepair sequence of the controlling sites, promoter and operator, for the lac operon.

Tryptophan Operon All 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

Two mechanisms of regulations for tryptophan operon # 1 Repressor/operator interaction with tryptophan as the effector molecule: Tryptophan binds the aporepressor (trp. R) and then binds operator to turn off the gene Power. Point® Layered Art

Two mechanisms of regulations for tryptophan operon # 2 Attenuation Control Regulatory Leader region Determines if initiated transcripts include other structural genes or not. Power. Point® Layered Art

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

# 2 Attenuation Control Absence of tryptophan or in the presence of low amounts of tryptophan: Under severe tryptophan starvation gene activity at maximum all long transcripts Under less severe situation gene Expression at less than maximum Long and short transcripts Greater the amount of tryptophan the greater the number of short transcripts Attenuation controls = > terminates transcription producing short transcripts m. RNA polymerase response to these m. RNA secondary structures

Transcription and translation are tightly coupled in prokaryotes Attenuation occurs at the m. RNA level and can reduce transcription of trp-operon 8 -or-10 fold. *Last long enough for Ribosome to load onto m. RNA. Position of ribosome on leader transcript determines if transcription is terminated or not. If starved for trytophan = lack trp-t. RNA If no trp-t. RNA, 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.

Starved for tryptophan Transcription continues ++ trptophan Termination signal For RNA polymerase, Which stops transcription

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 studying The regulation of the arabinose (ara) operon of E. coli. They found that instead of being regulated 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 the Polymerase could bind the operator and start transcription. In the ara operon, two molecules of Ara. C protein bind act as a bridge from the operator (ara. O 2 ) And to the promoter region ara I 1 which creates a loop that prevents the binding of CAP-c. AMP. With the addition of arabionose, the arabionose bound Ara. C protein is allosterically modified to bind to ara I 2, which allows CAP-c. AMP to bind the CAP site and positive regulate gene expression occurs.

However, for the ara operon to function, glucose can not be Present. If present, it will eliminate c. AMP and focus on the Utilization of glucose **Positive regulation of activators is now known to occur in a Variety of prokaryotic systems and in all eukaryotes.

Bacteriophage Gene regulation Regulation of gene expression in the lytic cycle and lysogeny in baceriophage lambda (λ) Excellent model for developmental switches in eukaryotic systems After λ infection of bacteria, a choice is made between lytic and lysogenic pathways 1) 2) Linear chromosome is circularized in host Transcription begins at PL & PR PL promoter for left early operon PR promoter for right early operon These promoters are on different DNA Strands.

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

c. I gene Cro gene N = protein is the antiterminator that allows RNA transcription past transcription terminator signals. Lysogeny Lytic c. I protein Integration of λ c. II protein stimulates synthesis of c. I repressor which competes with Cro protein. 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.

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