Gene Regulation & RNA Polymerases

• Gene regulation ensures cells respond to their environment and maintain their identity.

Short-term Gene Regulation

• Cells respond to extracellular environment, like growth factors and hormones.

Long-term Gene Regulation

• Tissue-specific identity of cells can be transmitted to daughter cells during division.

Gene Transcription

• The process by which information in a DNA strand is copied into an RNA molecule.
• RNA Polymerase enzymes catalyze this process.

RNA Polymerase Activity

• It adds nucleotides to the growing RNA chain.
• It catalyzes the formation of phosphodiester bonds between them.

Types of RNA Polymerases

• These are large multi-subunit enzymes.
• There are three RNA Polymerases in eukaryotes.
• RNA Pol I transcribes ribosomal RNA (rRNA)
• RNA Pol II transcribes mRNA and most non-coding RNAs
• RNA Pol III transcribes tRNA & other RNAs
• Prokaryotes use only one RNA Polymerase.

RNA Polymerase II

• It transcribes mRNA.
• It is a large multiprotein complex, consisting of 12 subunits (RBP1-12).
• RPB1, a DNA-directed RNA polymerase II subunit, catalyzes transcription of DNA to mRNA.
• Phosphorylation of RPB1 regulates enzyme activity.
• RNA Polymerase II cannot "read" DNA, requiring transcription factors for DNA binding and transcription.

RNA Polymerase II Transcription

• This process involves initiation, elongation, and termination.

Transcription Initiation

• RNA Pol II binds to DNA near the target gene at the core promoter, which is the transcription start site.
• RNA Pol II cannot "read" the DNA sequence to find where to bind.
• It is guided to the core promoter by proteins called transcription factors (TF).
• RNA Pol II and transcription factors form the pre-initiation complex.
• RNA Pol II phosphorylation at RBP1 initiates transcription.

Transcription Elongation

• RNA Pol II is released from the promoter.
• RNA Pol II unwinds the DNA into two separate strands called "transcription bubble".
• RNA Pol II moves along the DNA, using one strand as a template to synthesize a complementary RNA sequence.
• RNA Pol II adds nucleotides to the 3'-end of the growing RNA molecule.

Transcription Termination

• RNA Pol II encounters a termination signal where transcription stops.
• RNA Pol and mRNA are released.
• A new cycle of transcription begins.

Transcription Factors

• These are proteins that bind to specific DNA sequences and regulate (activate or inhibit) gene transcription.
• Most transcription factors do not work alone.
• These can recruit other proteins that activate (co-activators) or inhibit transcription (co-repressors).

Transcription Factors Structural Domains

• Functional units are shaped to allow a particular interaction (i.e. with DNA or other proteins).
• Structural Domains typically have:
• DNA-binding domain that recognizes specific short sequences near target gene.
• Trans-activation/trans-repression domain that contains binding sites for co-activators/co-repressors.
• Ligand-binding domain where some TFs require ligand binding, like hormone receptors for activation.
• Dimerization domain where some TFs need to form dimers to bind DNA.

DNA Binding Domains

• DNA binding domain shape allows them to bind to the major groove of DNA.
• They interact with nucleotide bases.
• The amino acid sequence of the DNA-binding domain determines the specific DNA sequences it can bind to.
• This determines TF specificity.
• A range of short (6-12bp) DNA sequences are recognized, called transcription factor binding motifs.
• TATA Binding protein binds to TATAAAA

Basic and Specific Transcription Factors

• Transcription factors are divided into two groups based on their mechanism of action.
• Basic Transcription Factors (only 6):
  • Found in all cells and tissues.
  • Recruit RNA Pol II to the gene promoter.
  • Transcriptional activators.
• Specific Transcription Factors (>2000):
  • Show tissue-specificity (e.g. heart-specific or lung-specific).
  • Regulate gene transcription.
  • Transcriptional activators or repressors.

Cis-Regulatory Elements

• Transcription factors bind to these elements.
• Core promoter and enhancer/silencer elements are regions of non-coding DNA that regulate the transcription of nearby genes.
• These elements contain transcription binding motifs.
• These elements recruit transcription factors (transcription activators or inhibitors) to the target gene.

Core Promoter

• This is immediately upstream (before) the gene.
• It has a fixed position (60-120bp).
• It contains the transcription start site (+1).
• Consists of binding sites for basal transcription factors.
• Basal transcription factors recruit RNA Pol II to the gene.
• It is required for gene transcription.

Enhancers/Silencers

• These can be upstream (before) or downstream (after) the gene promoter.
• They can be near (100bp) or very far (Mbp) from the gene promoter.
• They contain binding motifs for specific transcription factors.
  • More activators relate to enhancer, more repressors relate to silencer.
• They regulate the activity of the core promoter (enhances or inhibits).

Basal Transcription Factors

• These guide RNA Pol II to the core promoter.
• They bind to specific binding motifs on the core promoter.
• Basal transcription factors (TFII) guide the recruitment of RNA Pol II to the promoter.
• This leads to formation of pre-initiation complex.
• There are six basal transcription factors: TFIIA, TFIIB, TFIID, TFIIF, TFIIE and TFIIH.
• TFII factors are multi-subunit complexes with a specific function during transcription initiation.

Core Promoter Motifs

• Core promoter contains DNA motifs required for basal transcription factor binding.
• The TATA Box is TATA(A/T)A(A/T).
• The Initiator (Inr) contains the start point and is Py2CAPy2.
• The Downstream promoter element (DPE) is (A/G)G(A/T)(T/C)(A/C).

TFIID

• This basal transcription factor recognizes the core promoter.
• It is a multiprotein complex comprising a TATA box-binding protein (TBP) and 13-14 TBP-associated factors (TAFs).
• TBP recognizes the TATA box element.
• TAFs recognize the Initiator element and the DPE.
• TFIID initiates the assembly of the pre-initiation complex.

Assembly of Basal Transcription Complexes

• TFIID recognizes the core promoter.
• TFIIA stabilizes TFIID-DNA binding.
• TFIIB interacts with TFIID and determines RNA Pol II position.
• TFIIF interacts with RNA Pol II and recruits it to the promoter.
• TFIIE recruits TFIIH.
• TFIIH unwinds the DNA and activates RNA Pol II by phosphorylation.

Enhancers/Silencers characteristics

• Are non-coding DNA sequences that regulate promoter activity.
• Activity is independent of orientation or distance from gene promoter.
• Contain high density of binding motifs for different types of specific transcription factors.
• Binding of many different transcription factors takes place.

Enhancers/Silencers Binding Motifs

• These contain multiple binding motifs.
• Examples include: CAAT Box (GGCCAATCT), GC Box (GGCGG), Octamer (ATTTGCAT), and E Box (CACGTG).

Transcription Factor Binding Motifs

• These determine enhancer activity.
• Enhancer/silencer activity depends on:
  • Type of binding motifs (Predom. activators: Enhancer, Predom. repressors: Silencer).
  • Number of binding motifs.

Enhancers

• These increase gene transcription.
• Transcription factors on enhancer elements activate gene transcription by bringing co-activators to the gene promoter.
• Co-activators increase gene transcription by:
• Opening chromatin (euchromatin), to allow binding of RNA Pol II.
• Use Histone acetyltransferases for Histone acetylation.
• Use Chromatin remodeling complexes for Nucleosome displacement.
• Recruiting RNA Pol II and basal TFs to the target gene promoter and stabilizing the pre-initiation complex.

Silencers

• These inhibit gene transcription.
• Transcription factors on silencer elements can inhibit gene transcription by:
• Recruiting co-repressors which induce chromatin condensation (heterochromatin) and inhibit transcription.
• Use Histone deacetylases for Histone de-acetylation.
• Binding to DNA and directly blocking RNA Pol II recruitment to the target gene.

Chromosome Organization in the Nucleus

• Chromosomes at interphase occupy specific spaces, called chromosomal territories.
• Chromosomes are arranged radially around the nucleus and attached to specific sites on the nuclear envelope.
• Each chromosome is separated into a transcriptionally inactive (heterochromatic) and active (euchromatic) compartment.
  • The inactive compartment is close to the nuclear membrane.
  • The active compartment is within the nuclear interior.
• Each compartment is folded into loops called topological associated domains (TADs) that ensure enhancers are in close proximity to gene promoters.
• Each chromosome occupies a distinct region in the nucleus.
• They are separated into a transcriptionally active and inactive compartment
• Each compartment involves a number of TAD loops that ensure close proximity of enhancers to gene promoters.
• Each TAD includes chromatin loops that allow direct interaction of enhancers with promoters.
• Chromatin loops allow enhancers/silencers to interact with promoters.
  • The non-histone protein complex Cohesin binds to DNA forming a loop.
  • This enables the enhancer/silencer and promoter to come to be in very close proximity.

Cell Signalling Pathways

• These regulate transcription.
• Extracellular signals, such as growth factors and hormones, regulate gene transcription by activating cell signalling pathways.
• Specific transcription factors (activators or repressors) are recruited to gene promoters/enhancers.
• Activation or inhibition of gene transcription takes place.
• Transient changes in gene expression occur, with the effect lost when the stimulation stops.

Transcription Factors Combinations

• Specific transcription factors work in combinations to regulate transcription.
• Each gene is regulated by many different specific transcription factors (combinatorial regulation).
• It uses both activators and repressors.
• The cell integrates the effects of all transcription factors to switch gene transcription on or off.
• The cell switches according to stimuli (e.g. hormones, growth factors).
• The level of gene expression is determined according to the cell's requirements.
• Transcription factors and RNA Pol are held together and regulated by a multi-protein complex called the Mediator complex.

Rapid Gene Switching

• Cells can rapidly switch multiple genes on/off.
• Cells need to be able to rapidly switch multiple genes on or off at the same time.
• One transcription factor can regulate multiple genes.
• Cortisol is released under conditions of starvation and binds to the glucocorticoid receptor in liver cells.
• This simultaneously activates genes that increase blood glucose.
• In the absence of cortisol, glucose metabolism gene expression returns to normal levels.

Drugs Targeting Gene Transcription

• Drugs that target gene transcription play a key role in treating various diseases by normalizing the expression of dysregulated genes.
• Action is modulated by:
  • Receptor modulators.
  • Activate/inhibit receptors that regulate gene transcription.
  • Example is Tamoxifen (estrogen receptor modulator used in breast cancer).
  • Example is Dexamethasone (glucocorticoid receptor activator used in inflammatory diseases).
  • Transcription factor modulators.
  • Activate/inhibit transcription factors.
  • Example is Idasanutlin (p53 activator in clinical trials for different cancers).
• Histone deacetylase inhibitors.
  • Inhibit histone deacetylases – increase histone acetylation.
  • Example is Vorinostat (T cell lymphoma).
• RNA Polymerase inhibitors.
  • Inhibit RNA polymerase.
  • Example is Dactinomycin (chemotherapeutic for a number of cancers).