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Lec 3 

Lec 3 

Lecture 3: The control of Gene Expression

Gene:

  • The entire nucleic acid sequence that is necessary for the synthesis of functional gene product
  • This also includes genes upstream and downstream control sequences whether adjacent to the gene or not

Gene Families

  • Genes can be:
  • Solitary
  • Functionally grouped
  • Belong to gene families
  • In higher eukaryotes 25- 50% of genes are represented as solitary since the genome contains only one copy of them
  • Other genes are duplicated  and have close but non- identical sequences (high degree of homology)
  • Genes replicates in ‘cis’ like this are considered gene families 
  • Some families consist of hundreds of members (olfactory receptors) that have slightly different properties

Promotors Reminder

  • Upstream ‘switch’ for gene you want to express (GOI) – gene of interest
  • The Tata Caat and Gaca boxes are usually (but not always) part of what we call the core region (all usually within 100bps of start)
  • The Proximal region where (usually) numerous transcription factors bind
  • The distal region may or may not have control elements in it (control elements are more likely if we are dealing with a eukaryotic promotor)
  • Already aware that eukaryotic genes contain transcribed and non- transcribed regions known as exons and introns, respectively. (median intron length is 3.3 Kb)
  • Eukaryotic genes have more regulatory sequences – enhancers and silencers- than prokaryotic
  • Enhancers/ silencers are where activators and/ or repressors bind
  • Prokaryotic genes are typically found as polycistronic operons
  • A cistron is a unit coding for a single polypeptide
  • They are read through as a continuous pre- mRNA
  • A cluster of genes like this under the control of a single promotor is called an operon and is a single transcription unit


The operon

Organism development requires gene expression

  • cells need to express, different genes at different times in different places with an organism
  • Gene expression is needed to:
  • Allow developmental progression
  • Induce tissue differentiation
  • Induce a response to environmental stimuli
  • i.e. to make decisions
  • as a mechanism which can be acted on through Natural selection to modulate organism adaptation

Basics

  • for the most part the regulation of gene expression occurs or the level of transcription, where the production of hnRNA or mRNA from a gene is controlled
  • The lac operon is and example of gene expression at the level of transcription
  • However, gene expression can also be at the level of translation , where the production of a fully functional protein is controlled at any one of a number of stages
  • The Trp operon is and example of gene expression at the level of translation

The Lac Operon

  • The most famous gene regulation model
  • Francois Jacob and Jacques Monod in 1962
  • Many other prokaryotic genes are regulated in a similar fashion
  • The basic principles carry over into eukaryotes
  • An operon is a cluster of genes under the control of a single promotor
  • The lac operon codes for:
  • Enzymes involved in the degradation of lactose

Gene expression can allow ‘choices’ to be made by an organism

  • Bacteria need carbon – this is often utilised in sugar form
  • What if there are a number of different sugars in the environment?
  • Do the bacteria ‘prioritize’ which carbon source to use?
  • The answer is yes
  • Glucose is a carbon resource that can enter directly into glycolysis without being biochemically modified – and at no extra cost to the cell
  • Many other carbon resources have to be chemically modified to allow their use. This applies for example to all carbohydrate polymers including the sugar dimers
  • When given a ‘choice’, glucose or any carbon source that can directly enter into a catabolic pathway will be chosen over one which requires a greater energy input – this is clearly advantageous to the organism.. Why?
  • When that directly utilisable carbon source runs out (glucose), other slightly more complex sources of carbon can be used (such as lactose)
  • Lactose metabolism is governed by the Lac operon and is an example of controlled gene expression at the level of DNA
  • It allows bacteria to ‘choose’ between glucose and lactose (different resources) by ‘sensing’ the type and concentration of that resource in the environment
  • How is that the case?

The Lac operon Layout

  • Consists of 3 genes necessary for the metabolism of lactose
  • β-Galactosidase:
  • This enzyme hydrolyses lactose into glucose and galactose
  • Lactose permease:
  • Located in cell membrane and transfers lactose into the cell
  • Thiogalactoside transacetylase:
  • (transfers an acetyl group from acetyl-CoA to β-galactosides and is believed to have a role in cell detoxification)


  • 4 possible situations
  • When glucose is present and lactose is absent
  • The bacteria does not produce β- galactosidase
  • When glucose is present and lactose is present
  • The bacteria does not produce β- galactosidase
  • When glucose is absent and lactose is absent
  • The bacteria does not produce β- galactosidase
  • When glucose is absent and lactose is present
  • The bacteria does produce β- galactosidase

When lactose is absent the bacteria does not produce β- galactosidase

  • A repressor protein is continuously synthesised (constitutive expression)
  • It sits on a sequence of DNA just in front of the lac operon called the operator site when lactose is not present
  • The repressor protein lies just downstream of the promote site where RNA polymerase binds before it starts transcribing


When lactose is present polycistronic mRNA is produced

  • A small amount of allolactose is formed in the cell in the presence of lactose.
  • Allolactose binds the repressor protein at an allosteric site causing it to change its shape (a conformational change).
  • It can no longer sit on the operator site so RNA polymerase can now reach its promoter site


How is the operon NOT transcribed when both sugars are present?

  • When glucose and lactose are present RNA polymerase can sit on the promoter site but it is unstable and it keeps falling off
  • This is because a stabilising activator protein (called Catabolite Activator Protein (CAP) is missing!
  • This activator protein only works when glucose is absent


What happens when there is only lactose

  • In reality when there is only lactose present, The activator protein stabilizes RNA polymerase on the promoter site.
  • In this way E. coli only makes enzymes to metabolise other sugars in the absence of glucose


How does the Catabolite Activator Protein work?

  • When the glucose levels in the cell are high, the level of ATP is high and the cAMP level is low (and vice versa when the glucose levels are low)
  • cAMP combines with the CAP protein to form a CAP-cAMP complex that binds to part of the lac operon promoter.
  • This complex bends the DNA in a way that makes it much easier for RNA polymerase to bind to the promoter. Thus activating transcription, but only if the lac repressor isn’t present.
  • This is an example of positive regulation

Summary


Tryptophan (Trp) Operon

  • Tryptophan or Trp is an essential amino acid required for protein production. In the absence of a ready source of this amino acid, then Trp must be made from scratch.
  • Therefore the Trp operon is constitutively expressed unlike the Lac operon which is an activated (inducible) system.
  • So how is the Trp Operon switched off when there is an adequate supply of tryptophan?


  • The Trp promoter contains an operator sequence (a binding site for a repressor). It lies between 5 genes downstream and 1 upstream.
  • The repressor gene lies upstream of the Trp promoter and like the 5 genes involved in synthesis is also constitutively produced
  • Under normal conditions where trp is not present, the repressor does not bind the operator sequence and the genes are read and transcribed


  • When trp is present it binds the tetrameric repressor proteins and cause them to change shape.
  • In this form they can bind the Trp Operator and block transcription
  • The Trp Operon is therefore a repressible system

Attenuation

  • Control of the Trp Operon also happens at the level of Translation
  • The mechanism is called Attenuation 
  • This is a mechanism made possible because in prokaryotes translation begins before transcription finishes (no nuclear envelope).
  • The initial transcript termed “the leader sequence” contains 4 sequence-domains which are part-complimentary and can form hairpin loops
  • Thus the 4 domains 1,2, 3 and 4 can form the hairpins 1-2 and 3-4 or 2-3. It is clear all cannot be formed at the same time.
  • 3-4 is a termination hairpin > terminates translation.
  • The leader sequence (> 100 bases long) codes for a leader peptide about 14 aa’s long. At the beginning of this peptide two trp amino acids are coded for in a row. This also happens to be within Domain 1 of the hairpin-loop sequences.
  • It is highly unusual for two Tryptophan codons to be found together as trp is a rare amino acid, making this section acutely sensitive to the concentration of intracellular trp-tRNA.
  • If there is not enough charged trp-tRNA’s (low level of trp) available, then the ribosome will stall here and allow hairpin loop 2-3 enough time to form. This allows read-through of the mRNA
  • If there is no delay at this point (thus there is a high level of trp present) then the ribosome will NOT stall and instead allow hairpin loop 3-4 to form - this will terminate translation


Result

  • The Trp repressor decreases transcription approximately 70-fold in the presence of adequate concentrations of Tryptophan
  • The Attenuation of translation responds to the concentration of activated trp-tRNA’s and can decrease production of the protein product by a further 10-fold giving a 700 fold overall scale of control over tryptophan expression!!!!

Arabinose induction in the pGLO plasmid

  • Promoter called “pBAD”after products
  • The ara C gene encodes the regulator protein
  • There are 2 promoters reading in opposite directions:
  • pBAD and Pc
  • There are two operators in front of each promoter
  • I1 , I2 and O2 .
  • At its topmost level, The entire operon is under the control of glucose via CAP/CAMP and is not transcribed unless the glucose concentration is low

In the absence of Arabinose…

  • The AraC protein binds to the I1 and O2 operator sites & dimerises.
  • The looped DNA can neither bind the RNA Pol, nor the CAP/CAMP complex.
  • Arabinose is not necessary for dimerization here….


When arabinose is present…

  • Arabinose binds AraC which allows it to dimerise while bound to I1 and I2 .
  • At this point, the CAP-CAMP complex (if present) binds the CAP-binding site
  • Transcription occurs
  • Glucose must also be low in order for transcription to occur

Inducible versus repressible…

  • If transcription is activated (induced) or shut-down (repressed) when the switch is flicked (i.e. when the causative agent is added)
  • Inducible system:
  • Inducer required to bind to the promoter/operator then this is positive
  • Inducer required to release from promoter operator system then this is negative
  • Repressible system: This is called co-activation
  • Repressor required to bind to the promoter/operator then this is negative
  • Repressor required to release from promoter operator system then this is positive

Other Inducible/repressible systems of note..

  • INDUCIBLE
  • Neg Inducible = pLac promoter
  • Neg Inducible = pBAD promoter
  • Positive inducible = Tet-on (the activator rTTA needs the presence of Tetracycline to activate the gene downstream)
  • REPRESSIBLE
  • Pos repressible = Tet off promoter (The Repressor tTA binds to the promoter allowing transcription to proceed. Addition of Tetracyline binds tTA and removes it from DNA switching the gene off
  • So what is the Tryptophan system then?

Eukaryotic promoters

  • Very diverse
  • Typically lie upstream of the GOI
  • Often have regulatory elements several kilobases away from the transcriptional start site. Due to this placement, the transcriptional complex can cause the DNA to loop on itself.
  • Thus physically distant sections of DNA can impact gene expression
  • Distant regulators are called “enhancers” or “silencers”

Lec 3 

Lec 3 

Lecture 3: The control of Gene Expression

Gene:

  • The entire nucleic acid sequence that is necessary for the synthesis of functional gene product
  • This also includes genes upstream and downstream control sequences whether adjacent to the gene or not

Gene Families

  • Genes can be:
  • Solitary
  • Functionally grouped
  • Belong to gene families
  • In higher eukaryotes 25- 50% of genes are represented as solitary since the genome contains only one copy of them
  • Other genes are duplicated  and have close but non- identical sequences (high degree of homology)
  • Genes replicates in ‘cis’ like this are considered gene families 
  • Some families consist of hundreds of members (olfactory receptors) that have slightly different properties

Promotors Reminder

  • Upstream ‘switch’ for gene you want to express (GOI) – gene of interest
  • The Tata Caat and Gaca boxes are usually (but not always) part of what we call the core region (all usually within 100bps of start)
  • The Proximal region where (usually) numerous transcription factors bind
  • The distal region may or may not have control elements in it (control elements are more likely if we are dealing with a eukaryotic promotor)
  • Already aware that eukaryotic genes contain transcribed and non- transcribed regions known as exons and introns, respectively. (median intron length is 3.3 Kb)
  • Eukaryotic genes have more regulatory sequences – enhancers and silencers- than prokaryotic
  • Enhancers/ silencers are where activators and/ or repressors bind
  • Prokaryotic genes are typically found as polycistronic operons
  • A cistron is a unit coding for a single polypeptide
  • They are read through as a continuous pre- mRNA
  • A cluster of genes like this under the control of a single promotor is called an operon and is a single transcription unit


The operon

Organism development requires gene expression

  • cells need to express, different genes at different times in different places with an organism
  • Gene expression is needed to:
  • Allow developmental progression
  • Induce tissue differentiation
  • Induce a response to environmental stimuli
  • i.e. to make decisions
  • as a mechanism which can be acted on through Natural selection to modulate organism adaptation

Basics

  • for the most part the regulation of gene expression occurs or the level of transcription, where the production of hnRNA or mRNA from a gene is controlled
  • The lac operon is and example of gene expression at the level of transcription
  • However, gene expression can also be at the level of translation , where the production of a fully functional protein is controlled at any one of a number of stages
  • The Trp operon is and example of gene expression at the level of translation

The Lac Operon

  • The most famous gene regulation model
  • Francois Jacob and Jacques Monod in 1962
  • Many other prokaryotic genes are regulated in a similar fashion
  • The basic principles carry over into eukaryotes
  • An operon is a cluster of genes under the control of a single promotor
  • The lac operon codes for:
  • Enzymes involved in the degradation of lactose

Gene expression can allow ‘choices’ to be made by an organism

  • Bacteria need carbon – this is often utilised in sugar form
  • What if there are a number of different sugars in the environment?
  • Do the bacteria ‘prioritize’ which carbon source to use?
  • The answer is yes
  • Glucose is a carbon resource that can enter directly into glycolysis without being biochemically modified – and at no extra cost to the cell
  • Many other carbon resources have to be chemically modified to allow their use. This applies for example to all carbohydrate polymers including the sugar dimers
  • When given a ‘choice’, glucose or any carbon source that can directly enter into a catabolic pathway will be chosen over one which requires a greater energy input – this is clearly advantageous to the organism.. Why?
  • When that directly utilisable carbon source runs out (glucose), other slightly more complex sources of carbon can be used (such as lactose)
  • Lactose metabolism is governed by the Lac operon and is an example of controlled gene expression at the level of DNA
  • It allows bacteria to ‘choose’ between glucose and lactose (different resources) by ‘sensing’ the type and concentration of that resource in the environment
  • How is that the case?

The Lac operon Layout

  • Consists of 3 genes necessary for the metabolism of lactose
  • β-Galactosidase:
  • This enzyme hydrolyses lactose into glucose and galactose
  • Lactose permease:
  • Located in cell membrane and transfers lactose into the cell
  • Thiogalactoside transacetylase:
  • (transfers an acetyl group from acetyl-CoA to β-galactosides and is believed to have a role in cell detoxification)


  • 4 possible situations
  • When glucose is present and lactose is absent
  • The bacteria does not produce β- galactosidase
  • When glucose is present and lactose is present
  • The bacteria does not produce β- galactosidase
  • When glucose is absent and lactose is absent
  • The bacteria does not produce β- galactosidase
  • When glucose is absent and lactose is present
  • The bacteria does produce β- galactosidase

When lactose is absent the bacteria does not produce β- galactosidase

  • A repressor protein is continuously synthesised (constitutive expression)
  • It sits on a sequence of DNA just in front of the lac operon called the operator site when lactose is not present
  • The repressor protein lies just downstream of the promote site where RNA polymerase binds before it starts transcribing


When lactose is present polycistronic mRNA is produced

  • A small amount of allolactose is formed in the cell in the presence of lactose.
  • Allolactose binds the repressor protein at an allosteric site causing it to change its shape (a conformational change).
  • It can no longer sit on the operator site so RNA polymerase can now reach its promoter site


How is the operon NOT transcribed when both sugars are present?

  • When glucose and lactose are present RNA polymerase can sit on the promoter site but it is unstable and it keeps falling off
  • This is because a stabilising activator protein (called Catabolite Activator Protein (CAP) is missing!
  • This activator protein only works when glucose is absent


What happens when there is only lactose

  • In reality when there is only lactose present, The activator protein stabilizes RNA polymerase on the promoter site.
  • In this way E. coli only makes enzymes to metabolise other sugars in the absence of glucose


How does the Catabolite Activator Protein work?

  • When the glucose levels in the cell are high, the level of ATP is high and the cAMP level is low (and vice versa when the glucose levels are low)
  • cAMP combines with the CAP protein to form a CAP-cAMP complex that binds to part of the lac operon promoter.
  • This complex bends the DNA in a way that makes it much easier for RNA polymerase to bind to the promoter. Thus activating transcription, but only if the lac repressor isn’t present.
  • This is an example of positive regulation

Summary


Tryptophan (Trp) Operon

  • Tryptophan or Trp is an essential amino acid required for protein production. In the absence of a ready source of this amino acid, then Trp must be made from scratch.
  • Therefore the Trp operon is constitutively expressed unlike the Lac operon which is an activated (inducible) system.
  • So how is the Trp Operon switched off when there is an adequate supply of tryptophan?


  • The Trp promoter contains an operator sequence (a binding site for a repressor). It lies between 5 genes downstream and 1 upstream.
  • The repressor gene lies upstream of the Trp promoter and like the 5 genes involved in synthesis is also constitutively produced
  • Under normal conditions where trp is not present, the repressor does not bind the operator sequence and the genes are read and transcribed


  • When trp is present it binds the tetrameric repressor proteins and cause them to change shape.
  • In this form they can bind the Trp Operator and block transcription
  • The Trp Operon is therefore a repressible system

Attenuation

  • Control of the Trp Operon also happens at the level of Translation
  • The mechanism is called Attenuation 
  • This is a mechanism made possible because in prokaryotes translation begins before transcription finishes (no nuclear envelope).
  • The initial transcript termed “the leader sequence” contains 4 sequence-domains which are part-complimentary and can form hairpin loops
  • Thus the 4 domains 1,2, 3 and 4 can form the hairpins 1-2 and 3-4 or 2-3. It is clear all cannot be formed at the same time.
  • 3-4 is a termination hairpin > terminates translation.
  • The leader sequence (> 100 bases long) codes for a leader peptide about 14 aa’s long. At the beginning of this peptide two trp amino acids are coded for in a row. This also happens to be within Domain 1 of the hairpin-loop sequences.
  • It is highly unusual for two Tryptophan codons to be found together as trp is a rare amino acid, making this section acutely sensitive to the concentration of intracellular trp-tRNA.
  • If there is not enough charged trp-tRNA’s (low level of trp) available, then the ribosome will stall here and allow hairpin loop 2-3 enough time to form. This allows read-through of the mRNA
  • If there is no delay at this point (thus there is a high level of trp present) then the ribosome will NOT stall and instead allow hairpin loop 3-4 to form - this will terminate translation


Result

  • The Trp repressor decreases transcription approximately 70-fold in the presence of adequate concentrations of Tryptophan
  • The Attenuation of translation responds to the concentration of activated trp-tRNA’s and can decrease production of the protein product by a further 10-fold giving a 700 fold overall scale of control over tryptophan expression!!!!

Arabinose induction in the pGLO plasmid

  • Promoter called “pBAD”after products
  • The ara C gene encodes the regulator protein
  • There are 2 promoters reading in opposite directions:
  • pBAD and Pc
  • There are two operators in front of each promoter
  • I1 , I2 and O2 .
  • At its topmost level, The entire operon is under the control of glucose via CAP/CAMP and is not transcribed unless the glucose concentration is low

In the absence of Arabinose…

  • The AraC protein binds to the I1 and O2 operator sites & dimerises.
  • The looped DNA can neither bind the RNA Pol, nor the CAP/CAMP complex.
  • Arabinose is not necessary for dimerization here….


When arabinose is present…

  • Arabinose binds AraC which allows it to dimerise while bound to I1 and I2 .
  • At this point, the CAP-CAMP complex (if present) binds the CAP-binding site
  • Transcription occurs
  • Glucose must also be low in order for transcription to occur

Inducible versus repressible…

  • If transcription is activated (induced) or shut-down (repressed) when the switch is flicked (i.e. when the causative agent is added)
  • Inducible system:
  • Inducer required to bind to the promoter/operator then this is positive
  • Inducer required to release from promoter operator system then this is negative
  • Repressible system: This is called co-activation
  • Repressor required to bind to the promoter/operator then this is negative
  • Repressor required to release from promoter operator system then this is positive

Other Inducible/repressible systems of note..

  • INDUCIBLE
  • Neg Inducible = pLac promoter
  • Neg Inducible = pBAD promoter
  • Positive inducible = Tet-on (the activator rTTA needs the presence of Tetracycline to activate the gene downstream)
  • REPRESSIBLE
  • Pos repressible = Tet off promoter (The Repressor tTA binds to the promoter allowing transcription to proceed. Addition of Tetracyline binds tTA and removes it from DNA switching the gene off
  • So what is the Tryptophan system then?

Eukaryotic promoters

  • Very diverse
  • Typically lie upstream of the GOI
  • Often have regulatory elements several kilobases away from the transcriptional start site. Due to this placement, the transcriptional complex can cause the DNA to loop on itself.
  • Thus physically distant sections of DNA can impact gene expression
  • Distant regulators are called “enhancers” or “silencers”