Eukaryotic Gene Transcription: Molecular Mechanisms and Specificity Control

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February 5, 2025

Introduction

In this lecture, we are addressing the topic of eukaryotic gene transcription. Last time, we described the general elements and key components in the transcription initiation process. Today, we will delve deeper into the molecular aspects and establish the foundation for understanding the molecular mechanisms underlying the control of gene expression specificity in eukaryotic organisms, particularly multicellular organisms. We previously discussed the characteristics of the core and mentioned the existence of that recognize the core . These factors enable the correct positioning of the polymerase complex for all polymerases. We are primarily focusing on slides related to because it is the most studied and characterized, but the transcriptional logic is similar for and . We discussed the basal transcription complex or , which must form at the core to correctly position polymerase and define the direction of transcription. Some in the basal transcription complex help recognize the template strand and distinguish it from the coding strand. The combination of and forms the basal transcription complex or , a necessary key event. However, it is not sufficient by itself to specifically control gene transcription qualitatively and quantitatively. In other words, this complex is necessary but does not guarantee active gene transcription.

Pre-initiation Complex and General Transcription Factors

Preinitiation Complex Formation

The , necessary to initiate transcription, assembles through a four-step kinetic process:

Preinitiation Complex Assembly: The initial assembly of the complex. We will detail its components shortly.
Transcription Initiation: This phase is reminiscent of abortive transcription in prokaryotes but differs. Eukaryotic initiation requires ATP consumption due to the accessory activity of that denature downstream of the transcription start site.
DNA Denaturation and Kinase Activity: An activity is needed to denature near the transcription start site. Kinase activity is also essential, consuming ATP. This kinase activity is exerted by on , specifically on the Carboxyl-Terminal Domain () of .
Promoter Clearance and Elongation: Phosphorylation of the by , particularly , causes the holoenzyme complex to detach from the . This initiates clearance and the elongation phase. This is similar to the detachment of bacterial polymerase holoenzyme from the sigma subunit. The remaining proteins stay at the core or detach to start another transcription cycle.

General Transcription Factors (GTFs)

Several are required to initiate transcription. Their number varies across eukaryotes; humans have seven. The enzyme complex has 12 subunits, and the , bridging proximal elements and s to the core , is a multiprotein complex with many subunits. A significant portion of the coding genome is dedicated to proteins regulating transcription, besides . It’s estimated that humans have 3,000 transcription-related proteins, including , specific s, coactivators, and corepressors.

Schematic representation of Preinitiation Complex Formation. (Note: Image placeholder, replace with actual image if available).

Figure 1 shows the kinetic formation of the . An early key event is recognition by Binding Protein (). The associates with -associated factors (), some with transcriptional coactivator activity, like histone acetylation. and form the complex. assembly seems to be the first step in formation. Subsequently, accessory factors and join. helps define the transcription direction, distinguishing the template from the coding strand to orient polymerase processivity.

TF2D and TATA-Binding Protein (TBP)

The complex, comprising and , initiates assembly. recognizes and binds to the , a sequence in many gene s.

TF2B and Transcription Direction

, associating after , defines the transcription direction. It discriminates between template and coding strands, orienting polymerase. binding is asymmetric relative to the symmetry center of binding. binding to the bends . binds on one side, interacting with and recognizing the sequence, a GC-rich sequence upstream of the . Binding to the in the major groove and partially to the minor groove downstream of creates asymmetry, defining transcription direction. polymerase processivity is 5’ to 3’, so asymmetry defines the 3’ to 5’ template strand. thus defines template and coding strands through interaction.

TF2H: Helicase, Kinase, and Role in DNA Repair

is a crucial with helicase and kinase activities. Its helicase activity denatures the core, essential for transcription initiation, attributed to subunits and . Mutations in and genes cause , a rare genetic disorder with extreme sun sensitivity and high skin cancer risk.

Besides helicase activity, has kinase activity, ATP-dependent, used partly for helicase function and crucially for phosphorylation of . phosphorylation is essential for efficient transcription, transitioning from initiation to elongation. Without it, transcription stalls at initiation. Phosphorylation signals processing steps. For example, 5’ capping enzymes recognize phosphorylated , as do intron splicing complexes.

CTD Phosphorylation and RNA Processing

phosphorylation by is critical for denaturation, elongation, and coordinating processing. Phosphorylated is a platform for processing factors like capping, splicing, and termination enzymes.

The of has repeats of the consensus heptapeptide sequence with the consensus Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Repeat number varies; mammals have 52. Serine residues (Ser2 and Ser5, presumably) are phosphorylated by .

(Electrophoretic Mobility Shift Assay) demonstrates phosphorylation importance. assesses protein- complex molecular weight or formation changes.

Electrophoretic Mobility Shift Assay (EMSA): EMSA studies protein- interactions using native gel electrophoresis. Labeled with a protein-binding site is incubated with protein or extract. Protein binding retards migration in the gel, indicating interaction.

Procedure:

Prepare Labeled DNA Probe: Label fragment with sequence of interest.
Incubation: Incubate labeled with protein sample.
Gel Electrophoresis: Load mixture on non-denaturing polyacrylamide gel and perform electrophoresis.
Detection: Detect labeled . Unbound migrates faster; protein- complexes migrate slower (shifted band).
Supershift Assay (Optional): Add antibody specific to protein of interest to confirm protein identity. Antibody binding causes further mobility shift (supershift).

In the lecture’s experiment, a fragment containing a core is used with various (, , , , , ) and , with/without ATP. It tests if ATP is needed for molecular weight change in the multiprotein complex, indicating phosphorylation by . Results show a further mobility shift with and ATP, demonstrating ’s ATP-dependent phosphorylation, altering the complex.

factors and are also in , a sub-pathway repairing lesions in transcribed genes. Lesions like pyrimidine dimers and 6-4 photoproducts from UV radiation are repaired by . During transcription, , especially and , is crucial in . Inactivating mutations in these genes cause , increasing skin cancer and UV-related pathology risks like psoriasis. Patients must avoid sunlight.

Animation of Transcription Preinitiation Complex Assembly. (Note: GIF placeholder, replace with actual animation if available).

Figure 2 illustrates dynamic assembly, showing , , , , and other factor interactions.

Transcription by RNA Polymerases I and III

formation at the core isn’t exclusive to genes. and use similar recruitment at their gene s. differ, but transcriptional logic is analogous. is conserved across s of all three polymerases. and use different , like and for , and , , and for . Functional regulatory schemes are similar.

RNA Polymerase I (): Transcribes ribosomal genes ( in nucleolus. include (Upstream Binding Factor) and (Selectivity Factor 1). contains and , like for . s have a core element and upstream control element (UCE).
RNA Polymerase III (): Transcribes transfer ( genes, 5S rRNA genes, and other small genes. include , , and (for 5S rRNA genes). s can be downstream (internal s) or upstream of the start site.

Despite and sequence differences, formation with is conserved, indicating a common evolutionary origin for eukaryotic transcription initiation machinery.

Regulation of Transcription Specificity in Eukaryotes

Basal transcription complex formation is necessary but insufficient for effective gene transcription, resulting only in basal transcription, like weak s and basal bacterial polymerase transcription. Specific s are needed for quantitative and qualitative transcription control. These and cis-acting sequences like s and proximal s determine gene transcription timing and amount.

Basal transcription complex association with specific s (transcriptional activators) and the complex forms the activated transcription complex, often called the enhansiosome, essential for regulated gene expression.

Schematic representation of Activated Transcription Complex (Enhansiosome).

Figure 3 illustrates the activated transcription complex. Effective eukaryotic transcription initiation involves:

and polymerase attachment.
complex recruitment.
Chromatin remodeling complex and histone acetyltransferase association with specific transcriptional activators.

Event order varies. Sometimes, specific transcriptional activators bind first, then basal complex recruitment. Other times, the basal complex is present, and specific activator binding to s and proximal s initiates transcription via interaction.

Activated Transcription and Specific Transcription Factors

Specific s, or transcriptional activators, bind cis-regulatory elements like proximal s and s to modulate gene transcription. Crucial for tissue-specific and developmental gene expression, they work in concert with basal machinery and for precise gene activity control.

Cis-Regulatory Elements: Proximal Promoters and Enhancers

Cis-regulatory elements are sequences regulating genes on the same molecule. Proximal s and s are major eukaryotic cis-regulatory elements.

Distinguishing Features of Promoters and Enhancers

Proximal s are located within approximately 200 nucleotides upstream of the transcription start site, recognized by activators, with three key distinctions from s:

Distance-dependent: Activity depends on distance from start site.
Orientation-dependent: Function is sensitive to orientation relative to start site. Orientation change alters activity.
Position-dependent: Activity depends on position relative to transcription unit. Position shifts drastically affect transcription.

Enhancers, conversely, are farther, upstream, downstream, or intronic. They are opposite in characteristics:

Distance-independent: Function effectively far from .
Orientation-independent: Activity largely unaffected by orientation changes.
Position-independent: Function in different positions relative to , with limits.

This difference is due to and action. s, often distant, loop to approach s via the .

Proximal Promoter Elements: CAT box and GC box

Proximal s have characteristic motifs for specific activators. Two key elements are and .

CAT box: motif with consensus, -75 to -80 upstream of start site. Recognized by s like , , and .
GC box: GC-rich, often in proximal s, associated with . Recognized by activator . are regions with high cytosine-guanine dinucleotides, often in s, for gene regulation.

These functional elements were found using mutagenesis and assays.

Effect of Mutations in Promoter Regions on Transcriptional Activity.

Figure 4 shows such an experiment’s results. The graph shows transcriptional activity versus mutation positions upstream of the start site. Activity drops sharply at , , and positions, indicating their importance.

Enhancers: Structure and Enhansiosome Formation

s are positive regulatory elements enhancing transcription and contributing to tissue-specific gene expression. They are characterized by being composed of multiple binding sites for various transcriptional activators.

Structure of an Enhancer with Multiple Transcription Factor Binding Sites.

Figure 5 shows a representative , with multiple, often overlapping, binding sites for s like AP-1, AP-2, AP-3, AP-4, and . High regulatory element density is key in s, contrasting with spaced sites in proximal s.

s form the , activator complexes on the sequence. Activators in the assemble cooperatively, forming a loop to bring the near the . Activators in s include , , and .

Enhansiosome Formation and Interaction with Promoter.

Figure 6 depicts the with activators bound to the . They interact, loop , and bring the near the , activating transcription.

Gene Reporter Assays for Studying Regulatory Sequences

assays are powerful for studying regulatory sequences and transcriptional control, quantitatively measuring transcription driven by specific s or s.

Principle and Applications of Reporter Genes

A reporter gene has an easily detectable and quantifiable product. Common s:

$\beta$-galactosidase (lacZ gene): Activity measured using chromogenic substrates like , producing blue color upon cleavage.
(luc gene): Produces bioluminescence, measured with a luminometer.
Chloramphenicol Acetyltransferase (CAT): Activity measured by enzymatic assays.
Green Fluorescent Protein (GFP): Fluorescence measured directly via fluorometer or microscopy.

In assays, the regulatory sequence ( or ) is cloned upstream of the in a plasmid. This is transfected into cells. expression reflects regulatory sequence activity.

Figure 7 shows a assay design. Regulatory region cloned upstream of , transfected into cells. expression is measured to assess regulatory region activity.

Deletion Analysis: Maps regulatory regions using plasmids with progressively shorter regulatory fragments cloned upstream of the . Comparing expression in transfected cells identifies essential regions. Reduced activity upon deletion indicates important regulatory elements.

Mutagenesis: For finer mapping, mutagenesis introduces small, defined mutations (insertions or substitutions) sequentially across the regulatory region. Mutation effect on expression is assessed. Regions where mutations reduce activity are critical regulatory elements.

Analyzing Tissue-Specific Gene Expression

assays also study tissue-specific gene expression. Using regulatory sequences from tissue-specific genes and introducing constructs into different cell types or tissues (transgenic animals or embryo injection) determines regulatory sequence tissue specificity. For example, a responsive to Wnt/$\beta$-catenin signaling can drive a construct transfected into embryos. Spatial expression (e.g., $\beta$-galactosidase staining) identifies active tissues, like mesencephalon, vertebral precursors, or notochord in early development.

assays can probe gene regulation in disease models. In a mouse colon cancer model with APC tumor suppressor mutation, assays study specific activation in tumor vs. normal cells, identifying pathways and regulatory elements in tumorigenesis.

Mediator Complex: Bridging Activators and Basal Machinery

The complex, a large multiprotein complex, is vital in transcriptional regulation, bridging activators on s and proximal s to the basal transcriptional machinery at the core .

It consists of numerous proteins, combinatorially assembled, selectively including/excluding proteins gene-specifically. Typically >20 proteins, conserved phylogenetically, it bridges proteins and activators on cis sequences (s and proximal s). It also interacts with chromatin remodelers for chromatin structure regulation, including epigenetic functions.

According to the recruitment model, eukaryotic activator function is to stimulate polymerase binding to the core . An alternative model suggests activators induce transcriptional complex changes to boost efficiency. Evidence supports both models. However, understanding the exact role of eukaryotic activators may not be crucial for grasping their biological role.

Transcriptional Activators: Structure, Function, and Proto-oncogenes

We now discuss proteins involved in transcriptional regulation, focusing on specific s and briefly on the complex. These proteins, acting in trans, regulate transcription by binding to cis-regulatory elements like proximal s and s.

The complex, a large assembly of over 20 proteins (in humans and yeast), acts as a bridge between s and the basal transcriptional machinery. It is phylogenetically conserved and allows for combinatorial assembly, with subunit composition varying gene-specifically. While the recruitment model posits that activators enhance polymerase binding to the core , an alternative model suggests they modify the transcriptional complex to improve efficiency. Both models have supporting evidence, and the precise mechanism may not be critical to understanding their biological role.

Transcriptional activators are key to tissue-specific gene regulation. Their tissue-specific expression or activation allows them to control gene transcription by interacting with ubiquitously present genomic sequences. While major contributors to specificity, epigenetic mechanisms, non-coding s, and processing also play roles. Activators exhibit structural modularity, with functional domains often reused in combination. Notably, many are s. Their combinatorial action on s and proximal s, which have diverse architectures, enables the spatiotemporal control of gene expression in eukaryotes. The order of activator recruitment can be gene-specific, and chromatin structure and histone modifications further modulate their function.

General Characteristics and Tissue Specificity of Activators

Transcriptional activators mediate cellular responses by controlling gene expression. Examples include p53, Cb, Mic, Fos, Jun, $\beta$-catenin, and E2F. They respond to diverse cellular signals to regulate gene expression and cellular function. For instance, p53 responds to genotoxic and metabolic stress by regulating genes like (promoting apoptosis) and p21 (inducing cell cycle arrest). Cb mediates signals from G protein-coupled receptors, activating and , and is involved in hormone responses, such as thyroid hormone production upon stimulation. Growth factors like TGF-$\alpha$ and activate Fos through . Thus, activators translate diverse extracellular signals into specific changes in gene expression, achieving cell-type and tissue-type specific responses. We will discuss (inflammation, differentiation) and (embryonic development) in detail later.

Modular Structure of Transcriptional Activators

Transcriptional activators are structurally modular, featuring distinct domains for different functions. They typically have:

DNA Binding Domain (DBD): Recognizes specific sequences. This domain is well-structured, and activators are classified based on their structure.
Transcriptional Activation Domain (TAD): Interacts with the basal transcriptional machinery or the complex to stimulate transcription. The can be unstructured and flexible.
Linker Domain: A flexible domain connecting the and , providing structural independence and flexibility.

A common is the , found in phage lambda repressors, bacterial repressors, and . This motif uses a recognition $\alpha$-helix that inserts into the major groove to read the sequence. Activator families are classified by their structure, including:

()

These represent major families, with classification primarily based on structural features.

Combinatorial Control and Gene Regulation

Transcriptional activators function combinatorially. A limited set of regulatory elements and activators can generate vast regulatory diversity through different combinations. This combinatorial control allows for:

Differential Gene Regulation: Regulating diverse genes with varied functions.
Coordinated Gene Regulation: Regulating genes with shared functions in a coordinated manner.

Approximately 1800 s are known in humans, with about 400 specific to the 200 cell types, regulating an estimated 400,000 regions and 70,000 regions.

The Phantom 5 project demonstrated that cell types with similar phenotypes utilize s with similar characteristics. This highlights thatcell-type specific gene expression.

Transcriptional Activators as Proto-oncogenes and Oncogenes

Many transcriptional activators are s. s are normal genes controlling cell proliferation and survival. They can be converted into s by mutations or altered expression, leading to uncontrolled cell growth and cancer. This conversion can occur through:

Qualitative Changes: Mutations that alter the protein’s function, leading to hyperactivation or constitutive activation.
Quantitative Changes: Alterations in expression levels, such as increased transcription due to viral promoter insertion, chromosomal translocation near strong s, or gene amplification.

Examples of Oncogenic Activation:

c-Myc in Burkitt’s Lymphoma: In , translocation of the MYC from chromosome 8 to chromosome 14 places it under the control of the heavy chain . This results in constitutive overexpression of Myc in B lymphocytes, driving uncontrolled proliferation and development.
P53 Tumor Suppressor: p53 is a critical tumor suppressor, often mutated in human cancers. It is constitutively transcribed and translated but is kept at low levels by MDM2, an ubiquitin ligase that targets p53 for proteasomal degradation. damage and other cellular stresses inhibit MDM2, leading to p53 stabilization and accumulation. p53 then activates target genes involved in cell cycle arrest (p21) and apoptosis () to prevent tumor formation. p53 also regulates MDM2 expression, creating a negative feedback loop.

Clinical Relevance of Transcriptional Activators in Cancer:

Mutations in p53 are the most frequent genetic alterations in human tumors. Mutations in the of p53 can lead to loss of tumor suppressor function and, in some cases, gain of oncogenic function. These "oncomorphic" mutations can alter p53’s target gene specificity, shifting its role from tumor suppression to promoting tumorigenesis. Other transcriptional activators implicated in cancer include AP-1 (amplification, overexpression in tumors), (mutations in lymphoid tumors, autoimmune diseases), and (developmental disorders, ). These examples underscore the critical role of transcriptional activators in both normal physiology and human disease.

On Monday, we will explore the molecular mechanisms controlling the activity of these transcriptional activators to achieve cell-type and tissue-specific gene expression.

Conclusion

In summary, today’s lecture expanded on the fundamental mechanisms of eukaryotic transcription, focusing on the intricate interplay of the pre-initiation complex, general transcription factors, and regulatory cis-elements such as proximal s and s. We explored how the complex bridges transcriptional activators to the basal machinery, enabling precise control over gene expression. We also highlighted the modular structure of transcriptional activators and their combinatorial action in achieving tissue specificity. Finally, we discussed the critical role of transcriptional activators as s and their implications in human diseases, particularly cancer, with examples like MYC in and the multifaceted functions of p53.

Key Takeaways:

The Pre-initiation Complex (PIC) formation is a multi-step process essential for transcription initiation, involving and Polymerase.
TFIIH plays dual roles in transcription initiation and repair, linking these fundamental cellular processes.
Proximal s and s are cis-regulatory elements with distinct properties that dictate the specificity and level of gene transcription.
Gene reporter assays are powerful tools to dissect regulatory sequences and study tissue-specific gene expression.
The Mediator complex acts as a crucial interface between transcriptional activators and the basal transcription machinery.
Transcriptional activators are modular proteins with distinct domains for binding and transcriptional activation, allowing for combinatorial control of gene expression.
Many transcriptional activators are s, and their dysregulation is implicated in various human diseases, including cancer.

Further Questions to Consider:

How do chromatin remodeling complexes and histone modifications cooperate with transcriptional activators to regulate gene expression?
What are the specific molecular mechanisms by which different transcriptional activator families (, , , ) recognize and bind to ?
How do non-coding s contribute to the regulation of transcription specificity in eukaryotes?
What are the therapeutic strategies targeting transcriptional misregulation in cancer and other human diseases?

These questions will guide our future discussions and explorations into the fascinating world of eukaryotic gene regulation.

Thank you for your attention. Have a good weekend, and see you on Monday.

--- title: "Eukaryotic Gene Transcription: Molecular Mechanisms and Specificity Control" author: "Your Name" date: "2025-02-05" format: html: toc: true # Table of Contents toc-depth: 2 code-tools: true theme: cosmo # Or "journal" for Distill-like minimalism --- # Introduction In this lecture, we are addressing the topic of eukaryotic gene transcription. Last time, we described the general elements and key components in the transcription initiation process. Today, we will delve deeper into the molecular aspects and establish the foundation for understanding the molecular mechanisms underlying the control of gene expression specificity in eukaryotic organisms, particularly multicellular organisms. We previously discussed the characteristics of the core and mentioned the existence of that recognize the core . These factors enable the correct positioning of the polymerase complex for all polymerases. We are primarily focusing on slides related to because it is the most studied and characterized, but the transcriptional logic is similar for and . We discussed the basal transcription complex or , which must form at the core to correctly position polymerase and define the direction of transcription. Some in the basal transcription complex help recognize the template strand and distinguish it from the coding strand. The combination of and forms the basal transcription complex or , a necessary key event. However, it is not sufficient by itself to specifically control gene transcription qualitatively and quantitatively. In other words, this complex is necessary but does not guarantee active gene transcription. # Pre-initiation Complex and General Transcription Factors ## Preinitiation Complex Formation The , necessary to initiate transcription, assembles through a four-step kinetic process: 1. **Preinitiation Complex Assembly:** The initial assembly of the complex. We will detail its components shortly. 2. **Transcription Initiation:** This phase is reminiscent of abortive transcription in prokaryotes but differs. Eukaryotic initiation requires **ATP consumption** due to the accessory activity of that denature downstream of the transcription start site. 3. **DNA Denaturation and Kinase Activity:** An activity is needed to denature near the transcription start site. **Kinase activity** is also essential, consuming ATP. This kinase activity is exerted by on , specifically on the Carboxyl-Terminal Domain () of . 4. **Promoter Clearance and Elongation:** Phosphorylation of the by , particularly , causes the holoenzyme complex to detach from the . This initiates **clearance** and the **elongation phase**. This is similar to the detachment of bacterial polymerase holoenzyme from the sigma subunit. The remaining proteins stay at the core or detach to start another transcription cycle. ## General Transcription Factors (GTFs) Several are required to initiate transcription. Their number varies across eukaryotes; humans have seven. The enzyme complex has 12 subunits, and the , bridging proximal elements and s to the core , is a multiprotein complex with many subunits. A significant portion of the coding genome is dedicated to proteins regulating transcription, besides . It's estimated that humans have 3,000 transcription-related proteins, including , specific s, coactivators, and corepressors. ![Schematic representation of Preinitiation Complex Formation. (Note: Image placeholder, replace with actual image if available).](PreinitiationComplexFormation.png){#fig:PIC_formation_2 width="70%"} Figure [1](#fig:PIC_formation_2){reference-type="ref" reference="fig:PIC_formation_2"} shows the kinetic formation of the . An early key event is recognition by Binding Protein (). The associates with -associated factors (), some with transcriptional coactivator activity, like histone acetylation. and form the complex. assembly seems to be the first step in formation. Subsequently, accessory factors and join. helps define the transcription direction, distinguishing the template from the coding strand to orient polymerase processivity. ### TF2D and TATA-Binding Protein (TBP) The complex, comprising and , initiates assembly. recognizes and binds to the , a sequence in many gene s. ### TF2B and Transcription Direction , associating after , defines the transcription direction. It discriminates between template and coding strands, orienting polymerase. binding is asymmetric relative to the symmetry center of binding. binding to the bends . binds on one side, interacting with and recognizing the sequence, a GC-rich sequence upstream of the . Binding to the in the major groove and partially to the minor groove downstream of creates asymmetry, defining transcription direction. polymerase processivity is 5' to 3', so asymmetry defines the 3' to 5' template strand. thus defines template and coding strands through interaction. ### TF2H: Helicase, Kinase, and Role in DNA Repair is a crucial with helicase and kinase activities. Its **helicase activity** denatures the core, essential for transcription initiation, attributed to subunits and . Mutations in and genes cause , a rare genetic disorder with extreme sun sensitivity and high skin cancer risk. Besides helicase activity, has **kinase activity**, ATP-dependent, used partly for helicase function and crucially for phosphorylation of . phosphorylation is essential for efficient transcription, transitioning from initiation to elongation. Without it, transcription stalls at initiation. Phosphorylation signals processing steps. For example, 5' capping enzymes recognize phosphorylated , as do intron splicing complexes. ## CTD Phosphorylation and RNA Processing phosphorylation by is critical for denaturation, elongation, and coordinating processing. Phosphorylated is a platform for processing factors like capping, splicing, and termination enzymes. The of has repeats of the consensus heptapeptide sequence with the consensus `Tyr-Ser-Pro-Thr-Ser-Pro-Ser`. Repeat number varies; mammals have 52. Serine residues (Ser2 and Ser5, presumably) are phosphorylated by . (Electrophoretic Mobility Shift Assay) demonstrates phosphorylation importance. assesses protein- complex molecular weight or formation changes. ::: tcolorbox **Electrophoretic Mobility Shift Assay (EMSA):** EMSA studies protein- interactions using native gel electrophoresis. Labeled with a protein-binding site is incubated with protein or extract. Protein binding retards migration in the gel, indicating interaction. **Procedure:** 1. **Prepare Labeled DNA Probe:** Label fragment with sequence of interest. 2. **Incubation:** Incubate labeled with protein sample. 3. **Gel Electrophoresis:** Load mixture on non-denaturing polyacrylamide gel and perform electrophoresis. 4. **Detection:** Detect labeled . Unbound migrates faster; protein- complexes migrate slower (shifted band). 5. **Supershift Assay (Optional):** Add antibody specific to protein of interest to confirm protein identity. Antibody binding causes further mobility shift (supershift). ::: In the lecture's experiment, a fragment containing a core is used with various (, , , , , ) and , with/without ATP. It tests if ATP is needed for molecular weight change in the multiprotein complex, indicating phosphorylation by . Results show a further mobility shift with and ATP, demonstrating 's ATP-dependent phosphorylation, altering the complex. factors and are also in , a sub-pathway repairing lesions in transcribed genes. Lesions like pyrimidine dimers and 6-4 photoproducts from UV radiation are repaired by . During transcription, , especially and , is crucial in . Inactivating mutations in these genes cause , increasing skin cancer and UV-related pathology risks like psoriasis. Patients must avoid sunlight. ![Animation of Transcription Preinitiation Complex Assembly. (Note: GIF placeholder, replace with actual animation if available).](TranscriptionPreinitiationComplex.gif){#fig:PIC_animation_gif_2 width="70%"} Figure [2](#fig:PIC_animation_gif_2){reference-type="ref" reference="fig:PIC_animation_gif_2"} illustrates dynamic assembly, showing , , , , and other factor interactions. ## Transcription by RNA Polymerases I and III formation at the core isn't exclusive to genes. and use similar recruitment at their gene s. differ, but transcriptional logic is analogous. is conserved across s of all three polymerases. and use different , like and for , and , , and for . Functional regulatory schemes are similar. - **RNA Polymerase I ():** Transcribes ribosomal genes ( in nucleolus. include (Upstream Binding Factor) and (Selectivity Factor 1). contains and , like for . s have a core element and upstream control element (UCE). - **RNA Polymerase III ():** Transcribes transfer ( genes, 5S rRNA genes, and other small genes. include , , and (for 5S rRNA genes). s can be downstream (internal s) or upstream of the start site. Despite and sequence differences, formation with is conserved, indicating a common evolutionary origin for eukaryotic transcription initiation machinery. # Regulation of Transcription Specificity in Eukaryotes Basal transcription complex formation is necessary but insufficient for effective gene transcription, resulting only in basal transcription, like weak s and basal bacterial polymerase transcription. Specific s are needed for quantitative and qualitative transcription control. These and *cis*-acting sequences like s and proximal s determine gene transcription timing and amount. Basal transcription complex association with specific s (transcriptional activators) and the complex forms the **activated transcription complex**, often called the **enhansiosome**, essential for regulated gene expression. ![Schematic representation of Activated Transcription Complex (Enhansiosome).](ActivatedTranscriptionComplex.png){#fig:activated_complex_2 width="70%"} Figure [3](#fig:activated_complex_2){reference-type="ref" reference="fig:activated_complex_2"} illustrates the activated transcription complex. Effective eukaryotic transcription initiation involves: 1. and polymerase attachment. 2. complex recruitment. 3. Chromatin remodeling complex and histone acetyltransferase association with specific transcriptional activators. Event order varies. Sometimes, specific transcriptional activators bind first, then basal complex recruitment. Other times, the basal complex is present, and specific activator binding to s and proximal s initiates transcription via interaction. ## Activated Transcription and Specific Transcription Factors Specific s, or transcriptional activators, bind *cis*-regulatory elements like proximal s and s to modulate gene transcription. Crucial for tissue-specific and developmental gene expression, they work in concert with basal machinery and for precise gene activity control. ## Cis-Regulatory Elements: Proximal Promoters and Enhancers *Cis*-regulatory elements are sequences regulating genes on the same molecule. Proximal s and s are major eukaryotic *cis*-regulatory elements. ### Distinguishing Features of Promoters and Enhancers **Proximal s** are located within approximately 200 nucleotides upstream of the transcription start site, recognized by activators, with three key distinctions from s: - **Distance-dependent:** Activity depends on distance from start site. - **Orientation-dependent:** Function is sensitive to orientation relative to start site. Orientation change alters activity. - **Position-dependent:** Activity depends on position relative to transcription unit. Position shifts drastically affect transcription. **Enhancers**, conversely, are farther, upstream, downstream, or intronic. They are opposite in characteristics: - **Distance-independent:** Function effectively far from . - **Orientation-independent:** Activity largely unaffected by orientation changes. - **Position-independent:** Function in different positions relative to , with limits. This difference is due to and action. s, often distant, loop to approach s via the . ### Proximal Promoter Elements: CAT box and GC box Proximal s have characteristic motifs for specific activators. Two key elements are and . - **CAT box:** motif with consensus, -75 to -80 upstream of start site. Recognized by s like , , and . - **GC box:** GC-rich, often in proximal s, associated with . Recognized by activator . are regions with high cytosine-guanine dinucleotides, often in s, for gene regulation. These functional elements were found using mutagenesis and assays. ![Effect of Mutations in Promoter Regions on Transcriptional Activity.](PromoterActivityMutation.png){#fig:promoter_mutation_2 width="70%"} Figure [4](#fig:promoter_mutation_2){reference-type="ref" reference="fig:promoter_mutation_2"} shows such an experiment's results. The graph shows transcriptional activity versus mutation positions upstream of the start site. Activity drops sharply at , , and positions, indicating their importance. ### Enhancers: Structure and Enhansiosome Formation s are positive regulatory elements enhancing transcription and contributing to tissue-specific gene expression. They are characterized by being composed of multiple binding sites for various transcriptional activators. ![Structure of an Enhancer with Multiple Transcription Factor Binding Sites.](EnhancerStructure.png){#fig:enhancer_structure_2 width="70%"} Figure [5](#fig:enhancer_structure_2){reference-type="ref" reference="fig:enhancer_structure_2"} shows a representative , with multiple, often overlapping, binding sites for s like AP-1, AP-2, AP-3, AP-4, and . High regulatory element density is key in s, contrasting with spaced sites in proximal s. s form the , activator complexes on the sequence. Activators in the assemble cooperatively, forming a loop to bring the near the . Activators in s include , , and . ![Enhansiosome Formation and Interaction with Promoter.](EnhansiosomeFormation.png){#fig:enhansiosome_2 width="70%"} Figure [6](#fig:enhansiosome_2){reference-type="ref" reference="fig:enhansiosome_2"} depicts the with activators bound to the . They interact, loop , and bring the near the , activating transcription. ## Gene Reporter Assays for Studying Regulatory Sequences assays are powerful for studying regulatory sequences and transcriptional control, quantitatively measuring transcription driven by specific s or s. ### Principle and Applications of Reporter Genes A **reporter gene** has an easily detectable and quantifiable product. Common s: - **$\beta$-galactosidase (*lacZ* gene):** Activity measured using chromogenic substrates like , producing blue color upon cleavage. - **(*luc* gene):** Produces bioluminescence, measured with a luminometer. - **Chloramphenicol Acetyltransferase (CAT):** Activity measured by enzymatic assays. - **Green Fluorescent Protein (GFP):** Fluorescence measured directly via fluorometer or microscopy. In assays, the regulatory sequence ( or ) is cloned upstream of the in a plasmid. This is transfected into cells. expression reflects regulatory sequence activity. ![Schematic of a Reporter Gene Assay.](ReporterGeneAssay.png){#fig:reporter_assay_2 width="70%"} Figure [7](#fig:reporter_assay_2){reference-type="ref" reference="fig:reporter_assay_2"} shows a assay design. Regulatory region cloned upstream of , transfected into cells. expression is measured to assess regulatory region activity. **Deletion Analysis:** Maps regulatory regions using plasmids with progressively shorter regulatory fragments cloned upstream of the . Comparing expression in transfected cells identifies essential regions. Reduced activity upon deletion indicates important regulatory elements. **Mutagenesis:** For finer mapping, mutagenesis introduces small, defined mutations (insertions or substitutions) sequentially across the regulatory region. Mutation effect on expression is assessed. Regions where mutations reduce activity are critical regulatory elements. ### Analyzing Tissue-Specific Gene Expression assays also study tissue-specific gene expression. Using regulatory sequences from tissue-specific genes and introducing constructs into different cell types or tissues (transgenic animals or embryo injection) determines regulatory sequence tissue specificity. For example, a responsive to Wnt/$\beta$-catenin signaling can drive a construct transfected into embryos. Spatial expression (e.g., $\beta$-galactosidase staining) identifies active tissues, like mesencephalon, vertebral precursors, or notochord in early development. assays can probe gene regulation in disease models. In a mouse colon cancer model with *APC* tumor suppressor mutation, assays study specific activation in tumor vs. normal cells, identifying pathways and regulatory elements in tumorigenesis. ## Mediator Complex: Bridging Activators and Basal Machinery The complex, a large multiprotein complex, is vital in transcriptional regulation, bridging activators on s and proximal s to the basal transcriptional machinery at the core . It consists of numerous proteins, combinatorially assembled, selectively including/excluding proteins gene-specifically. Typically \>20 proteins, conserved phylogenetically, it bridges proteins and activators on *cis* sequences (s and proximal s). It also interacts with chromatin remodelers for chromatin structure regulation, including epigenetic functions. According to the recruitment model, eukaryotic activator function is to stimulate polymerase binding to the core . An alternative model suggests activators induce transcriptional complex changes to boost efficiency. Evidence supports both models. However, understanding the exact role of eukaryotic activators may not be crucial for grasping their biological role. ## Transcriptional Activators: Structure, Function, and Proto-oncogenes We now discuss proteins involved in transcriptional regulation, focusing on specific s and briefly on the complex. These proteins, acting in *trans*, regulate transcription by binding to *cis*-regulatory elements like proximal s and s. The complex, a large assembly of over 20 proteins (in humans and yeast), acts as a bridge between s and the basal transcriptional machinery. It is phylogenetically conserved and allows for combinatorial assembly, with subunit composition varying gene-specifically. While the recruitment model posits that activators enhance polymerase binding to the core , an alternative model suggests they modify the transcriptional complex to improve efficiency. Both models have supporting evidence, and the precise mechanism may not be critical to understanding their biological role. Transcriptional activators are key to tissue-specific gene regulation. Their tissue-specific expression or activation allows them to control gene transcription by interacting with ubiquitously present genomic sequences. While major contributors to specificity, epigenetic mechanisms, non-coding s, and processing also play roles. Activators exhibit structural modularity, with functional domains often reused in combination. Notably, many are s. Their combinatorial action on s and proximal s, which have diverse architectures, enables the spatiotemporal control of gene expression in eukaryotes. The order of activator recruitment can be gene-specific, and chromatin structure and histone modifications further modulate their function. ### General Characteristics and Tissue Specificity of Activators Transcriptional activators mediate cellular responses by controlling gene expression. Examples include p53, Cb, Mic, Fos, Jun, $\beta$-catenin, and E2F. They respond to diverse cellular signals to regulate gene expression and cellular function. For instance, p53 responds to genotoxic and metabolic stress by regulating genes like (promoting apoptosis) and p21 (inducing cell cycle arrest). Cb mediates signals from G protein-coupled receptors, activating and , and is involved in hormone responses, such as thyroid hormone production upon stimulation. Growth factors like TGF-$\alpha$ and activate Fos through . Thus, activators translate diverse extracellular signals into specific changes in gene expression, achieving cell-type and tissue-type specific responses. We will discuss (inflammation, differentiation) and (embryonic development) in detail later. ### Modular Structure of Transcriptional Activators Transcriptional activators are structurally modular, featuring distinct domains for different functions. They typically have: - **DNA Binding Domain (DBD):** Recognizes specific sequences. This domain is well-structured, and activators are classified based on their structure. - **Transcriptional Activation Domain (TAD):** Interacts with the basal transcriptional machinery or the complex to stimulate transcription. The can be unstructured and flexible. - **Linker Domain:** A flexible domain connecting the and , providing structural independence and flexibility. A common is the , found in phage lambda repressors, bacterial repressors, and . This motif uses a recognition $\alpha$-helix that inserts into the major groove to read the sequence. Activator families are classified by their structure, including: - - () - - These represent major families, with classification primarily based on structural features. ### Combinatorial Control and Gene Regulation Transcriptional activators function combinatorially. A limited set of regulatory elements and activators can generate vast regulatory diversity through different combinations. This combinatorial control allows for: - **Differential Gene Regulation:** Regulating diverse genes with varied functions. - **Coordinated Gene Regulation:** Regulating genes with shared functions in a coordinated manner. Approximately 1800 s are known in humans, with about 400 specific to the 200 cell types, regulating an estimated 400,000 regions and 70,000 regions. The Phantom 5 project demonstrated that cell types with similar phenotypes utilize s with similar characteristics. This highlights thatcell-type specific gene expression. ### Transcriptional Activators as Proto-oncogenes and Oncogenes Many transcriptional activators are s. s are normal genes controlling cell proliferation and survival. They can be converted into s by mutations or altered expression, leading to uncontrolled cell growth and cancer. This conversion can occur through: - **Qualitative Changes:** Mutations that alter the protein's function, leading to hyperactivation or constitutive activation. - **Quantitative Changes:** Alterations in expression levels, such as increased transcription due to viral promoter insertion, chromosomal translocation near strong s, or gene amplification. **Examples of Oncogenic Activation:** - **c-Myc in Burkitt's Lymphoma:** In , translocation of the *MYC* from chromosome 8 to chromosome 14 places it under the control of the heavy chain . This results in constitutive overexpression of Myc in B lymphocytes, driving uncontrolled proliferation and development. - **P53 Tumor Suppressor:** p53 is a critical tumor suppressor, often mutated in human cancers. It is constitutively transcribed and translated but is kept at low levels by MDM2, an ubiquitin ligase that targets p53 for proteasomal degradation. damage and other cellular stresses inhibit MDM2, leading to p53 stabilization and accumulation. p53 then activates target genes involved in cell cycle arrest (p21) and apoptosis () to prevent tumor formation. p53 also regulates MDM2 expression, creating a negative feedback loop. **Clinical Relevance of Transcriptional Activators in Cancer:** Mutations in p53 are the most frequent genetic alterations in human tumors. Mutations in the of p53 can lead to loss of tumor suppressor function and, in some cases, gain of oncogenic function. These \"oncomorphic\" mutations can alter p53's target gene specificity, shifting its role from tumor suppression to promoting tumorigenesis. Other transcriptional activators implicated in cancer include AP-1 (amplification, overexpression in tumors), (mutations in lymphoid tumors, autoimmune diseases), and (developmental disorders, ). These examples underscore the critical role of transcriptional activators in both normal physiology and human disease. On Monday, we will explore the molecular mechanisms controlling the activity of these transcriptional activators to achieve cell-type and tissue-specific gene expression. # Conclusion In summary, today's lecture expanded on the fundamental mechanisms of eukaryotic transcription, focusing on the intricate interplay of the pre-initiation complex, general transcription factors, and regulatory *cis*-elements such as proximal s and s. We explored how the complex bridges transcriptional activators to the basal machinery, enabling precise control over gene expression. We also highlighted the modular structure of transcriptional activators and their combinatorial action in achieving tissue specificity. Finally, we discussed the critical role of transcriptional activators as s and their implications in human diseases, particularly cancer, with examples like *MYC* in and the multifaceted functions of p53. ::: tcolorbox **Key Takeaways:** - The **Pre-initiation Complex (PIC)** formation is a multi-step process essential for transcription initiation, involving and Polymerase. - **TFIIH** plays dual roles in transcription initiation and repair, linking these fundamental cellular processes. - **Proximal s** and **s** are *cis*-regulatory elements with distinct properties that dictate the specificity and level of gene transcription. - **Gene reporter assays** are powerful tools to dissect regulatory sequences and study tissue-specific gene expression. - The **Mediator complex** acts as a crucial interface between transcriptional activators and the basal transcription machinery. - **Transcriptional activators** are modular proteins with distinct domains for binding and transcriptional activation, allowing for combinatorial control of gene expression. - Many transcriptional activators are **s**, and their dysregulation is implicated in various human diseases, including cancer. ::: ::: tcolorbox **Further Questions to Consider:** - How do chromatin remodeling complexes and histone modifications cooperate with transcriptional activators to regulate gene expression? - What are the specific molecular mechanisms by which different transcriptional activator families (, , , ) recognize and bind to ? - How do non-coding s contribute to the regulation of transcription specificity in eukaryotes? - What are the therapeutic strategies targeting transcriptional misregulation in cancer and other human diseases? ::: These questions will guide our future discussions and explorations into the fascinating world of eukaryotic gene regulation. Thank you for your attention. Have a good weekend, and see you on Monday.