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”