Molecular Biology - Gene Regulation Notes

Gene Regulation

• All cells in a multicellular organism possess the same set of genes.
• However, not all genes are expressed in every cell at all times.
• Different cell types (e.g., muscle, cornea, brain, pancreas) require different sets of genes to be expressed to perform their specific functions.
  • For example, insulin should only be produced in the pancreas, not in the brain.
• Within a specific cell type, certain genes are expressed only during particular periods.
  • For instance, genes involved in wound healing are expressed only upon injury and are repressed once healing is complete.
• Circadian clocks involve morning and evening genes that are periodically activated and deactivated each day.
• An organism's survival relies on its capacity to express or repress genes in varying combinations across different times and cell types.
• Understanding how a gene is activated when its product is needed and deactivated when it is no longer required is crucial.

Activators

• Activators are DNA-binding proteins that positively regulate gene expression.
• They function through two primary mechanisms:
  • Cooperative binding:
    • The activator simultaneously interacts with the promoter and RNA polymerase.
    • This interaction recruits the enzyme to the promoter, enhancing transcription from promoters that are limited by RNA polymerase binding.
  • Allostery:
    • The activator interacts with the closed complex where RNA polymerase is bound to the promoter.
    • This interaction induces conformational changes in either the promoter or the polymerase, leading to the formation of an open complex and the initiation of transcription.

Positive Regulation

• In positive regulation (or positive control), an activator protein must bind to activate transcription.
• Example: Catabolite Activator Protein (CAP) or cyclic AMP Response Protein (CRP) in E. coli.
  • CAP binds to the CAP binding site in the lac promoter and recruits RNA polymerase, thereby activating transcription of the lac operon.

Repressors

• Repressors are DNA-binding proteins that negatively regulate gene expression.
• The DNA binding site for a repressor is called an operator.
  • The operator overlaps with the RNA polymerase binding site in the promoter.
• When a repressor binds to the operator, it reduces or eliminates RNA polymerase binding to the promoter, thus repressing transcription.

Negative Regulation

• In negative regulation (or negative control), the binding of a repressor protein prevents transcription, and its removal is necessary for transcription to occur.
• Example: The lac repressor in E. coli binds to the lac operator.
  • This binding prevents RNA polymerase from binding to the lac promoter and initiating transcription of the lac operon.
• Lactose can remove the repressor from the promoter.
  • This allows RNA polymerase to bind and initiate transcription of the lac operon.

Co-repressor

• A co-repressor binds to an inactive repressor, converting it into an active repressor.

Structural Gene

• Any gene that codes for a protein.
• Examples: lacZ, lacY, and lacA.
  • These structural genes code for β-galactosidase, lactose permease, and β-galactoside transacetylase, respectively.

Regulator Gene

• A gene that codes for a protein involved in regulating other genes.
• Example: lacI, codes for the lac repressor, which regulates the lac operon.

Cis-Acting Sequences or Elements

• Regulatory DNA sequences linked to structural genes.
• They regulate only the genes physically linked to them.

Trans-Acting Products

• Trans-acting products (proteins) bind to cis elements to regulate gene expression.
• Examples:
  • RNA polymerase
  • Transcription factors
  • Repressors
  • Activators
• These protein products regulate the expression of genes that are not physically linked to them.

Lac Operon

• An operon in E. coli that contains three genes involved in lactose metabolism: lacZ, lacY, and lacA.

Components

• lacI: Regulatory gene
• lac P: lac I promoter
• P: promoter
• O: Operator
• Structural genes: lacZ, lacY, lacA, Galactoside permease, Thiogalactoside transacetylase

E. Coli Preference

• E. coli can utilize either glucose (a monosaccharide) or lactose (a disaccharide) for energy.
• Lactose requires hydrolysis (digestion) before it can be used.
• Therefore, E. coli preferentially uses glucose when available.

Four Situations are possible

1. Glucose present, lactose absent: E. coli does not produce β-galactosidase.
2. Glucose present, lactose present: E. coli does not produce β-galactosidase.
3. Glucose absent, lactose absent: E. coli does not produce β-galactosidase.
4. Glucose absent, lactose present: E. coli does produce β-galactosidase.

• Under normal conditions, the lac operon is 'turned off' or repressed because the lac repressor is always bound to the Olac.
• However, this binding is not indefinitely strong.
• Consequently, the lac operon is expressed at a very low basal level (about 5 molecules per cell).
• This low-level expression ensures that the cell has a minimum level of the necessary machinery to act upon lactose when it becomes available.
• When lactose is present, it is transported into the cell by lactose permease and converted into allolactose by β-galactosidase (which is expressed at the basal level).
• Allolactose binds to the lac repressor, causing a conformational change that prevents the repressor from binding to the Olac.

Lactose Absent

• A repressor protein is continuously synthesized.
• It binds to the operator site (DNA sequence just upstream of the lac operon).
• The repressor protein blocks the promoter site where RNA polymerase binds to initiate transcription.

Lactose Present

• A small amount of allolactose is formed within the bacterial cell.
• Allolactose binds to the repressor protein at an allosteric site.
• This binding causes a conformational change in the repressor protein.
• The altered repressor can no longer bind to the operator site.
• RNA polymerase can now access the promoter site and initiate transcription.

Both Glucose and Lactose Present

• RNA polymerase can bind to the promoter site, but the binding is unstable.
• RNA polymerase tends to fall off.

Glucose Absent and Lactose Present

• RNA polymerase has a weak binding affinity to the lac operon promoter.
• Stable binding is ensured by the formation and binding of a cyclic AMP (cAMP) and CAP complex.
• CAP only functions when glucose is absent.
• In this way, E. coli only produces enzymes to metabolize other sugars when glucose is unavailable.

Control of lac Operon by Glucose

• The lac operon's function is to break down lactose into galactose and glucose.
• Galactose is also eventually converted into glucose by the gal operon.
• An additional control mechanism ensures that the lac operon is turned off not only when lactose is absent but also when there is sufficient glucose.
• After lac operon induction, the концентрация of glucose increases in the cell.
• This increase in glucose concentration leads to a lower concentration of cAMP, which is necessary for the formation of the cAMP-CAP activator complex.
• As a result, the concentration of cAMP-CAP decreases, and the expression of the lac operon is reduced.
• This regulation is logical, as there is no need to uptake lactose and produce glucose from it when there is already an adequate amount of glucose.
• Therefore, even if lactose is added to the medium, the lac operon will not be activated if there is sufficient glucose in the cell.

Repression of the lac Operon

• The number of lac repressor molecules remains constant in the cell due to constitutive synthesis.
• Following lac operon induction, the number of lactose molecules decreases due to their breakdown by β-galactosidase.
• Eventually, all lactose is consumed, and it is no longer available to inactivate the repressor.
• Consequently, the repressor is free to bind to the operator and turn off the lac operon.
• This repression is logical because there is no need for the lac operon to be expressed when no lactose is present.

Trp Operon

• The trp operon is a group of genes that are transcribed together.
• It codes for the components required for the production of tryptophan.
• Example for Repressible operon
• The trp operon in E. coli comprises five genes (trpE, trpD, trpC, trpB, trpA).
• These genes encode three enzymes necessary for synthesizing tryptophan from chorismate.

Induction

• Tryptophan is an amino acid required for cellular processes, including protein translation and gene regulation (as a co-repressor of its own operon).
• When there is sufficient tryptophan in the cell, all trp aporepressor molecules are converted to active repressors, effectively repressing the trp operon.
• When tryptophan levels decrease, tryptophan is diverted for other cellular uses, reducing the amount available to act as a co-repressor.
• Consequently, the trp operon is induced until a sufficient quantity of tryptophan is produced.
• The trp operon is regulated (turned on and off) in response to the cellular concentration of tryptophan.

Negative Control

• The trp operon is also under negative control, similar to the lac operon.
• However, the lac operon codes for catabolic enzymes that break down lactose, whereas the trp operon codes for anabolic enzymes that synthesize tryptophan.
• Therefore, while the presence of lactose should induce the lac operon to break it down, the presence of tryptophan should repress the trp operon because there is no need for its synthesis.
• All genes of the trp operon are transcribed as a polycistronic mRNA from the trp promoter (Ptrp).
• E. coli has a regulator gene, trpR, which produces the trp aporepressor.
• Tryptophan functions as a co-repressor; it binds to the trp aporepressor, inducing a conformational change in the protein, converting it into an active repressor.
• The active trp repressor binds to the trp operator (Otrp), preventing transcription because Otrp overlaps with the RNA polymerase binding site in Ptrp.
• Therefore, the trp operon is said to be under negative regulation or negative control in the presence of tryptophan.

Control by Attenuator

• Attenuation is a secondary mechanism of negative feedback in the trp operon.
• While the trp repressor reduces transcription by a factor of 70, attenuation can further decrease it by a factor of 10, resulting in a cumulative repression of approximately 700-fold.
• Attenuation is possible because, in prokaryotes without a nucleus, ribosomes begin translating the mRNA while RNA polymerase is still transcribing the DNA sequence.
• This allows translation to directly affect transcription of the operon.

Leader Transcript (trpL)

• At the beginning of the transcribed genes of the trp operon, there is a sequence of 140 nucleotides called the leader transcript (trpL).
• This transcript includes four short sequences designated 1-4:
  • Sequence 1 is partially complementary to sequence 2.
  • Sequence 2 is partially complementary to sequence 3.
  • Sequence 3 is partially complementary to sequence 4.
• Three distinct secondary structures (hairpins) can form: 1-2, 2-3, or 3-4.
• The formation of the 1-2 structure prevents the formation of the 2-3 structure, while the formation of 2-3 prevents the formation of 3-4.
• The 3-4 structure is a transcription termination sequence.
  • Once it forms, RNA polymerase will dissociate from the DNA, and transcription of the structural genes will not occur.

Leader Peptide

• Part of the leader transcript codes for a short polypeptide of 14 amino acids, called the leader peptide.
• This peptide contains two adjacent tryptophan residues, which is rare because tryptophan is an uncommon amino acid.
• If the ribosome attempts to translate this peptide when tryptophan levels are low, it will stall at one of the two trp codons.
• While stalled, the ribosome physically shields sequence 1 of the transcript, preventing it from forming the 1-2 secondary structure.
• Sequence 2 is then free to hybridize with sequence 3, forming the 2-3 structure, which prevents the formation of the 3-4 termination hairpin.
  • The 2-3 structure is called the anti-termination hairpin.
• RNA polymerase is then free to continue transcribing the entire operon.
• If tryptophan levels are high, the ribosome will translate the entire leader peptide without interruption and will only stall during translation termination at the stop codon.
• At this point, the ribosome physically shields both sequences 1 and 2.
• Sequences 3 and 4 are then free to form the 3-4 structure, which terminates transcription.
• The outcome is that the operon will be transcribed only when tryptophan is unavailable for the ribosome, while the trpL transcript is constitutively expressed.

Gal Operon

• An operon in E. coli that contains three genes (galE, galT, galK) which code for three enzymes involved in galactose metabolism.
• Like the lac operon, the gal operon codes for catabolic enzymes; therefore, the presence of galactose should induce the expression of the gal operon.

Negative Control

• All genes of the gal operon are transcribed into a single polycistronic mRNA from the gal promoter (Pgal).
• E. coli has a regulator gene, galR, which produces the gal repressor.
• There are two operators for the gal repressor: galOe (at the 5’ end of the promoter) and galOi (located further downstream inside the galE gene).
• In the absence of galactose, the gal repressor binds to the gal operators (galOe and galOi), preventing transcription because the operator overlaps with the RNA polymerase binding site in the Pgal.
• Therefore, the gal operon is said to be under negative regulation or negative control.

Control by Glucose

• Glucose is more efficiently utilized than galactose as a carbon source.

• Therefore, additional mechanisms ensure that the gal operon is turned off not only in the absence of galactose but also when there is enough glucose.

• This control is achieved because glucose is produced from galactose by the action of the gal operon.

• After induction of the gal operon, the concentration of glucose in the cell increases.

• The increased glucose concentration decreases the concentration of cAMP, which is required to form the cAMP-CAP activator complex.

• As a result, the concentration of cAMP-CAP is lowered, and the expression of the gal operon is proportionately reduced.

• This is logical because there is no need to convert galactose to glucose when there is enough glucose already.

• Therefore, even if galactose is added to the medium, the gal operon is not turned on if there is sufficient glucose in the cell.

• However, UDP-galactose is also a precursor in the synthesis of the E. coli cell wall. Therefore, the cell must be capable of synthesizing UDP-galactose at all times.

• In the absence of endogenous galactose, UDP-glucose is converted to UDP-galactose by galactose epimerase (GalE).

• At that time, glucose should not negatively affect the expression of the gal operon.

• This is achieved through the promoters of the gal operon.

  • There are two gal promoters for the gal operon:
    • One initiates transcription at transcript start site G1
    • The other initiates transcription at G2.

• The G1 and G2 transcripts differ in length by 5 nucleotides but contain the entire three functional genes (galE, galT, galK).

  • The G1 transcript is more abundant and responsive to cAMP-CAP
    • This transcript is controlled by the glucose level.
  • The G2 transcript is less abundant and not responsive to cAMP-CAP.
    • This transcript is produced regardless of the glucose status of the cell.

• As a result, even though the gal operon level is substantially reduced via the control of the abundant G1 transcript by glucose, a sufficient level of this operon is maintained via the less abundant G2 transcript, which is not affected by glucose.

Ara Operon

• The ara operon codes for three enzymes that are required to catalyze the metabolism of arabinose.

Positive Regulation

If arabinose is present and glucose is absent:

• Arabinose binds to AraC protein.
• The AraC-arabinose complex binds to the araI site (the inducer site).
• cAMP-CAP binds to the CAP site.
• RNA polymerase binds the Ara promoter site (PBAD).
• The genes for arabinose enzymes B, A, and D are transcribed, translated, and arabinose is metabolized.

Negative Regulation

If arabinose is absent:

• AraC protein, without the inducer (arabinose), still binds to the araI site, but with a different result.
• AraC on the araI site helps other AraC protein molecules bind to the operator site (araO site).
  • When arabinose is present, AraC cannot bind to araO.
• AraC proteins on the araI and araO sites interact, bending the DNA into a loop that suppresses transcription.