Advanced Transcriptional Regulation: Motifs, Epigenetics, and Steroid Signaling
Overview of Transcriptional Regulation and Transcription Factors
Transcription factors are broadly categorized into specific families based upon the structural domains or motifs present within their protein sequences.
Regulation involves two primary types of factors and DNA elements:
Activators and Enhancers: Activators are regulatory transcription factors that bind to enhancer elements to increase the rate of transcription.
Repressors and Silencers: Repressors are regulatory transcription factors that bind to silencer elements to decrease or inhibit transcription.
These regulatory transcription factors interface with the general (basal) transcription machinery to control gene expression.
Mechanisms Linking General Machinery and Regulatory Factors
There is a physical connection between the general transcription machinery and regulatory factors, often mediated through a structure called the Mediator complex.
Transcription factor activity is modulated by several mechanisms:
Small Effector Molecules: These molecules can bind to transcription factors to regulate their ability to bind DNA or recruit other proteins.
Protein-Protein Interactions: Biologists study which proteins physically contact each other inside the cell with the assumption that physical contact implies a shared functional relationship.
Covalent Modifications: Transcription factors can be modified through processes like phosphorylation or methylation, which changes their activity levels.
DNA Methylation and the Biology of Aging
Methylation can occur on the DNA itself, rather than just on the proteins.
GC Islands (CpG Islands): These are regions located next to promoters. When these GC islands are methylated, it serves as a hard rule for transcriptional silencing.
For methylation in other genomic locations, there is no universal rule; the impact on activation or inhibition must be inspected on a case-by-case basis.
Global Methylation and Lifespan: As humans age, their DNA becomes more methylated globally. The speaker suggests this acts like a "program" restricting humanity to a specific age.
Lifespan Anecdote: While the biblical text suggests a limit of years, the speaker mentions claims of individuals living for or years due to radically different lifestyles and avoiding certain foods that affect them energetically.
Systems Biology vs. Single-Gene Studies
Regulation of a single gene typically involves a large number of transcription factors, not just one.
The Spark Plug Metaphor: Studying a single gene to make statements about an entire organism is compared to opening a car hood, looking at a single spark plug, and making comments about the entire engine.
Synergy: In a whole system, the interaction of parts creates synergy, where the result is more than the sum of individual parts.
Systems Biology: This field emerged to look at all components of an organism simultaneously rather than in isolation.
Protein Domains and Motifs in Transcription Factors
Domain: A region on a protein that, when folded in three dimensions, occupies a specific spatial location.
Motifs: Signature secondary structures within domains that appear frequently in different proteins.
Fractal Nature of Protein Structure:
Common secondary structures include the Alpha helix and Beta sheet.
The alpha helix resembles a vortex, a shape seen in tornadoes, hurricanes, the water going down a toilet, and galaxies.
Fractal Definition: The idea that laws governing large-scale structures (like hurricanes) also govern small-scale structures (like proteins).
Specific Transcription Factor Motifs
Helix-Turn-Helix (HTH): Found in the sigma factor; consists of an alpha helix, a short turn (3 to 4 amino acids), and another alpha helix.
Helix-Loop-Helix (HLH):
Function: A dimerization motif.
Structure: Two alpha helices connected by a loop. The loop is longer than a "turn."
Size: Approximately amino acids.
Mechanism: Allows two proteins to communicate and associate into a dimer.
Zinc Finger Motif:
Function: Helps the protein recognize and bind to the major groove of the DNA backbone.
Structure: Composed of one alpha helix and two antiparallel beta strands forming a beta sheet.
Coordination: Uses a divalent cation, Zinc (), held in place by coordination bonds with cysteine R-groups.
Leucine Zipper:
Function: Dimerization motif.
Structure: Two alpha helices wrap around each other to form a coiled-coil.
Mechanism: Leucine (single-letter designation L) R-groups are positioned on one side of an alpha helix. These nonpolar R-groups (structure: and two methyl groups) interlock like the teeth of a zipper with another protein chain.
Multi-Motif Factors: A single transcription factor may contain more than one type of motif.
Dimerization and Nature's Symmetry
Homodimer: A complex formed by two identical protein subunits.
Heterodimer: A complex formed by two different protein subunits.
Symmetry in Nature: Nature favors symmetry, as seen in palindromic restriction sites and the bilateral symmetry of the human body.
Properties of Enhancers and Silencers
Cis-acting elements: Like promoters and the lac operon CAP site, enhancers and silencers are located on the same DNA molecule as the gene they regulate.
Orientation Independent: They can be taken out and flipped degrees and still function.
Distance and Position: They can function at large distances (over base pairs away), can be located upstream or downstream of the promoter, and can even be found within introns.
Upregulation vs. Downregulation:
Upregulation: Increasing transcription rates, sometimes by -fold to -fold via enhancers.
Downregulation: Decreasing transcription rates via silencers.
The General Transcription Machinery: RNA Polymerase II
TFIID (Transcription Factor II D):
A general/basal transcription factor complex required for all protein-coding genes.
Contains the TATA box binding protein (TBP), which recognizes the TATA box.
RNA Polymerase II cannot bind the TATA box on its own and requires TFIID.
Coactivators:
Essential for increasing transcription but cannot bind DNA directly.
They work by contacting other transcription factors already bound to the DNA.
Trans Activation Domain: A region on the coactivator that contacts the basal machinery to increase the transcription rate.
Mediator and Phosphorylation:
Mediator bridges regulatory factors and basal machinery.
Hyperphosphorylation: The Carboxy Terminal Domain (CTD) of RNA Polymerase II must be hyperphosphorylated to transition from initiation to the elongation phase.
NASCAR Metaphor: Hyperphosphorylation is like the flag dropping in a NASCAR race, signaling the cars to go.
Kinases involved: TFIIH (Transcription Factor II H) and the kinase subunit of the Mediator complex provide the phosphate groups.
Regulation by Steroid Hormones
Steroid Hormones:
Derived from Cholesterol (characterized by four fused rings: three hexagons and one pentagon).
Property: Small and nonpolar, allowing them to pass directly through the plasma membrane.
Function: Produced in one gland, travel through the blood at low concentrations, and affect gene expression and behavior in target tissues.
Examples: Estrogen, Testosterone, and Glucocorticoids (involved in glucose metabolism).
Glucocorticoid Signaling Pathway:
The Glucocorticoid Receptor (GR) acts as a transcription factor but is often called a "receptor."
In the cytoplasm, the GR is held in an inactive monomeric state by a complex involving HSP90 (Heat Shock Protein ).
Binding of the hormone to the receptor triggers the release of HSP90 and subsequent steps in the genetic circuitry.
Experimental Analysis: Gel Shift Assay (EMSA)
Used to determine if a factor binds DNA and if that binding is dependent on a small effector like a hormone.
Setup for Hormone Dependence Study:
Probe Alone: Runs quickly to the bottom of the native polyacrylamide gel.
Probe + Transcription Factor: If no shift occurs, the factor cannot bind DNA alone.
Probe + Transcription Factor + Hormone: A shift (slower migration) indicates the hormone allows the factor to bind DNA, likely by changing its conformation.