Control of Gene Expression - Focus on Prokaryotes

Gene Expression

• Definition: It is the process by which information from a gene directs the synthesis of a gene product in the form of a protein or functional RNA molecule

  • As a result, gene expression can affect the phenotypes (or observable traits) of cells/organisms

Control of Gene Expression

• Specifically refers to the process of turning ON and OFF genes

• Crucial for giving different cells their various properties

  • In simplest form, the cells of an organism have the same DNA → turning ON and OFF parts of the genes allow for the correct genes to be expressed within different cells

Steps at Which Gene Expression is Regulated

• Initiation of Transcription

  • MAIN POINT OF CONTROL

    • Makes it very efficient b/c this prevents a lot of resources from being used to create gene products that aren’t even going to end up being used

  • Here, the cell decides whether to even begin copying a gene into RNA

  • If the “promoter” isn’t activated through the binding of RNA Polymerase and additional relevant “players,” nothing else happens

• RNA Processing (Splicing, Capping, Polyadenylation)

  • Through alternative splicing, a cell can create different proteins from the same gene by choosing which exons to keep in / leave out

  • By monitoring capping and polyadenylation, a cell can actually control which mRNA molecules are allowed to pass through the nuclear pore into the cytoplasm

• mRNA Degradation

  • The process in which mRNA strands are broken down in the nucleus can affect which signals end up getting “shut off” → Since they never make it to being translated into proteins

• Initiation of Translation

  • The cell can determine whether ribosomes are recruited to specific mRNA strands to build protein

• Protein Folding and Post-Translational Modification

  • Even if a protein is created, it doesn’t always remain “active”

  • The cell can often add groups (like phosphate) or clip pieces off to “activate” the protein

How Transcription Regulators/Factors Work

• Very important to differentiate them from general transcription factors

• Definition: These proteins bind to regulatory sequences (additional DNA elements) to control gene expression

• Process

  • Transcription regulator proteins interact with DNA through a DNA binding domain

    • “Transcription factor binding site (Response element/motif)”

      • Short DNA sequence that is recognized and bound by these proteins

  • More specifically, these proteins interact with DNA through contacts in the major and minor groove

Consensus Motif

• Refers to the common or “average” DNA sequence that is shared among many binding sites for the same transcription factor regulator

• In a way, it acts like a perfect blueprint or roadmap at a major/minor groove to compare REAL transcription regulator/factor binding sites too

  • In the consensus motif, there are the essential nucleotides, or the bases that never change

  • It shows the preference for certain nucleotides on it → Letter height is proportional to the preference for that nucleotide (Taller = more preferred)

• IMPORTANT: The closer the actual response motif sequence is to the preferred sequence (consensus motif), the more likely the transcription regulator will bind

Interactions with Major and Minor Grooves

• Major groove

  • Refers to the wider gap between the backbones (about 22 angstrom wide)

  • Leaves the “faces” of the nitrogenous bases more exposed

• Minor groove

  • Refers to the narrow gap (about 12 angstrom wide)

  • It is shallower and more cramped

• Process

  • Amino acid side chains make hydrogen, ionic, and hydrophobic bonds with nucleotide base pairs in the major (and minor) groove to “READ” the DNA

    • Typically, it is mainly the major groove because each base pair combination is very unique (a protein can tell the difference between A-T and T-A w/o unzipping the helix), and there’s more space to allow bulky proteins

  • Each of the four base pairs (A-T, T-A, G-C, C-G) have unique patterns of hydrogen bond donors and acceptors so that the transcription regulator can recognize a specific sequence in the DNA

Repressors versus Activators

• Repressors: Bind DNA to prevent RNA Polymerase from initiating transcription

• Activators

  • There are activation domains, or regions within a transcription regulator protein

  • These domains can promote the transcription of a gene by interacting with the transcription machinery

  • Can help promote expression from inefficient promoters

    • Allowing RNA Polymerase to associate more strongly with promoters

Operons + Operators

• Operons

  • Refer to clusters of genes that are co-regulated (or the transcription of these genes are simultaneously managed)

    • These clusters usually ultimately result in the synthesis of different proteins that are all responsible for a specific task within a specialized bacterial cell

  • They exist b/c of the polycistronic nature of bacterial cells

    • Which allow for multiple proteins to be synthesized from a single mRNA strand

• Operators

  • Regulatory DNA sequences in/near the promoter region (NOT -35/-10 sequences)

  • Its accessibility versus inaccessibility is what drives the initiation of transcription

    • Specifically, for many operons, the default setting is “OFF” as a transcription repressor is physically occupying the major groove of the operator

    • The removal of that repressor allows for the production of the entire team of enzymes to be made possible

Tryptophan operon

• In E.coli cells, the tryptophan (trp) operon is present in order for the regulation of the biosynthesis of the amino acid tryptophan to occur

• Specifically, E.coli cells can control the operon by sensing the concentration of tryptophan in the cell

  • Low levels of tryptophan

    • Causes the operon to be ON

• High levels of tryptophan

  • Causes the operon to be OFF

• Mechanisms behind this regulation

  • Trp repressor acts as an allosteric enzyme

    • Meaning its confirmation/shape can be changed according to whether binding occurs at the allosteric site of the repressor

    • Specifically, in cells with lower levels of tryptophan, the Trp repressor remains inactive, such that its shape is incompatible with binding to the operator

      • Makes the operator accessible → RNA Polymerase can bind at promoter site

    • On the other hand, in cells with higher levels of tryptophan, the Trp repressor has tryptophan bind to its allosteric site, causing the repressor to be active now as the change in confirmation permit DNA binding (to the operator)

      • Makes the operator inaccessible → RNA Polymerase can no longer bind at promoter site

    • Feedback inhibition

      • This is an example of feedback inhibition

        • B/c the abundance of the products (tryptophan) causes the product to bind to the allosteric enzyme and make it inactive

Lac operon

• Cluster of lac operon genes allows for the regulation of enzymes that breakdown lactose in bacteria

  • This is b/c lactose needs to be turned into the preferred food source in E. coli, glucose

  • And so, these operon genes code for different proteins that all play a role in this process

    • LacZ: Breaks down lactose to make glucose

    • LacY: Allows lactose to enter the bacterial cells

    • LacA: Promotes lactose breakdown

• Regulation mechanism

  • At lacO operator, LacI repressor (protein) can bind to it to block the transcription of the operon

    • This happens in the absence of lactose since there’s no need to create the enzymes when nothing needs to be broken down

  • In the presence of lactose

    • Allolactose (structural isomer of lactose) binds to the LacI repressor, causing it to dissociate from the lacO operator

      • Allowing RNA Polymerase to bind at promoter, permitting transcription

  • When there is A LOT of lactose, not enough glucose

    • Then, there really needs to be a need for these enzymes responsible for breaking down lactose to prevail

      • NEED LOTS OF GENE EXPRESSION!!

    • cAMP levels are high then

      • These levels that are high can just be understood as the necessary factors for CAP activator proteins to bind to the CAP site → PROMOTING RNA Polymerase transcription initiation