Lecture 7 (Kaufmann) Flashcards
Transcriptional regulation in eukaryotes
What are induced pluripotent stem (iPS) cells
iPS Cells
- Induced pluripotent stem cells are adult cells that have been genetically reprogrammed to revert to a pluripotent state, meaning they can develop into various cell types (e.g., blood, gut, cardiac cells)
Anatomy of the eukaryotic Nucleus
Eukaryotic Nucleus Structure
- Nucleus: Control center with DNA.
- Nuclear Envelope: Double membrane with pores for transport.
- Chromatin: DNA + proteins; euchromatin (active) and heterochromatin (inactive).
- Nucleolus: Ribosome production.
- Pores: Allow material exchange with cytoplasm.
Genome size and gene numbers in eukaryotic
genomes
Genome size and Gene Numbers of important Organisms
Structure of a Eukaryotic Gene
A Eukaryotic Gene
- Enhancer: distal control elements that interact with transcription factors –> far away from promotor
- Proximal Control Elements: interact with TF –> Close to promotor
- Promotor: RNA polymerase binds to initiate transcription
- Exons: Coding sequences of DNA
- Introns: Non-coding sequences that are spliced out of the pre-mRNA during RNA processing.
- Poly-A sequence: A signal in the mRNA that specifies the addition of a poly-A tail
- Termination region: signals the end of transcription
Role of Histones in the eukaryote evolution
Role of Histones in the eukaryote evolution
- Histones are Proteins that compact and organize DNA into chromatin, allowing efficient storage and regulation of genetic material.
- Compacted DNA facilitated by histones enables eukaryotes to store more genes compared to less compacted prokaryotic DNA.
- The ability to manage and store larger genomes contributed to the rise of eukaryotic cellular and organismal complexity.
- Symbiotic relationships, such as the incorporation of mitochondria (endosymbiosis), provided energy efficiency and supported genome expansion.
Promotor Structure in Eukaryotes
Promotor Structure in Eukaryotes
- Eukaryotic transcription is often under positive regulation (promoter is inactive unless it is bound by transcription factor(s))
- BREu (Upstream) and BREd (Downstream) are sequences recognized by the tf TFIIB
- TATA box (25-30 bp) is a binding site for the TATA-binding protein (TBP), a subunit of the transcription factor complex TFIID.
- The initiator (Inr) element encompasses the transcription start site (+1)
- IMR refers to motifs that might enhance transcription initiation by interacting with other components of the promoter.
RNA Polymerases in Eukaryotes
Three RNA polymerases in eukaryotes
RNA Polymerase I
- synthesizes rRNA in the nucleolus
RNA Polymerase II
- transcribes mRNA, snRNA and IncRNA (long non-coding RNAs) in the nucleoplasm
RNA Polymerase III
- transcribes tRNA, 5s rRNA in the nucleoplasm & other small RNA
The Promotor of RNA Polymerase I
The Promotor of RNA Polymerase I
- The RNA Pol I promoter consists of a core promoter and an upstream control element (UPE)
- The factor UBF1 warps DNA around a protein structure to
bring the core and UPE into proximity
- Core-binding factor (SL1) includes the factor TBP that is involved in initiation by all three RNA polymerases
- RNA polymerase binds to the UBF1-SL1 complex at the core
promoter
The Promotor of RNA Polymerase II
The Promotor of RNA Polymerase II
- RNA pol II requires general transcription factors to initiate
transcription
- RNA polymerase II promoters commonly have a short conserved sequence (initiator element Inr) at the start point
- The TATA box is a common component of RNA polymerase II
promoters and consists of an A-T-rich octamer located ~25 bp upstream of the start point
- The downstream promoter element (DPE) is a common component of RNA pol II promoters that do not contain a TATA box
- A core promoter for RNA pol II generally includes the InR and either TATA box or a DPE
The Promotor of RNA Polymerase III
The Promotor of RNA Polymerase III
- Internal promoters have short consensus sequences located within the transcription unit and cause initiation at a fixed distance upstream
- Upstream promoters can contain three short consensus sequences upstream of the startpoint that are bound by transcription factors
The Transcription Initiation
The Transcription Initiation
- RNA Polymerase II promoters include a TATA box or downstream promoter element (DPE), and an initiator element (Inr).
- TFIID binds to the TATA box via TBP domain (TATA-binding protein), bending the DNA and aiding promoter recognition.
- TFIIB bridges TFIID and RNA Polymerase II, positioning the polymerase at the start site.
- TFIIH unwinds the DNA (helicase activity) and phosphorylates the CTD of RNA Polymerase II to initiate transcription.
- RNA Polymerase II begins transcription, with CTD phosphorylation coupling transcription and RNA processing.
The CTD of RNA Polymerases II
The CTD of RNA Polymerases II
- The CTD of RNA Polymerase II contains amino acids which can be easily phosphorylated ( heptapeptide repeats).
- Phosphorylation by TFIIH kinase regulates transcription stages like initiation, elongation, and termination.
- CTD phosphorylation couples transcription with RNA processing and chromatin modification.
- Its low-complexity structure enables interactions and allows liquid–liquid phase separation to form transient compartments.
What are (potential) functions of CTD phosphorylation of RNA pol II?
Functions of CTD Phosphorylation of RNA Pol II:
Regulates Transcription Stages:
- Controls initiation, elongation, and termination of transcription.
Recruits mRNA Processing Enzymes:
- Capping enzyme binds the phosphorylated CTD
- SCAFs (SR-like CTD- associated factors) may bind the CTD and then recruit splicing factors
- Some components of cleavage/polyA also bind the CTD already after initiation of transcription
Liquid–Liquid Phase Separation:
- The Pol II CTD can form liquid phase-separated compartments, which means that instead of transcription factors (TFs) binding individually to the DNA, they can come together in a droplet-like compartment
Dynamic Modulation:
- Phosphorylation states change to coordinate transcription and RNA processing.
What is an Enhancer?
Enhancer
- Like the promoter, an enhancer is a modular element constructed of short DNA sequence elements that bind various types of transcription factors.
- An enhancer typically activates the promoter nearest to itself and can be any distance either upstream or downstream of the promoter.
- Enhancers form complexes of activators that interact directly or indirectly with the promoter.
Enhancer RNAs (eRNAs)
Enhancer RNAs (eRNAs)
- Enhancer RNAs may be predictive of active enhancers: eRNA transcription correlates with target gene transcription in inducible systems and in different cell types and often, though not always, precedes target gene activation
- Enhancers mainly produce short, unstable transcripts (bi-directional) starting at nucleosome-depleted regions (NDRs)
Upstream antisense RNAs
Upstream antisense RNAs
- Short RNA molecules transcribed from upstream promoter regions.
- These are transcribed by separate RNA Polymerase II (Pol II) complexes from transcription start sites (TSSs) that are oriented in the opposite (antisense) direction to the main gene.
- They are located at the upstream edge of the nucleosome-depleted region (NDR) in the proximal promoter, where transcription factor binding sites are present.
- Their role isn’t fully clear but may involve gene regulation, influencing transcriptional activity or chromatin structure.
Differences and commonalities of enhancer and promoter features
Differences and Similarities Between Enhancers and Promoters
Similarities
- Both enhancers and promoters initiate transcription by recruiting transcription factors and RNA Polymerase II.
- They are associated with nucleosome-depleted regions and produce transcripts.
Differences
- Enhancers act at a distance to boost gene transcription, producing unstable eRNAs, while promoters are located near the TSS and produce stable pre-mRNAs.
- Only promoter-driven transcripts are stabilized post-initiation by splice sites and the absence of premature polyadenylation signals.
- Enhancers regulate multiple genes, while promoters typically regulate the downstream gene.
Regulation of core promoter activation
Step 1
Step 1: PIC assembly and Pol II recruitment
- Transcription factors (TFs) and cofactors (COFs) help assemble the pre-initiation complex (PIC) at the core promoter.
- RNA Polymerase II is recruited to the transcription start site (TSS) by general transcription factors (TFIID, TFIIB, TFIIE, etc.).
- Enhancers can loop to interact with the promoter, boosting Pol II recruitment.
- Coactivators like CBP/p300 interact with the mediator complex, linking the enhanceosome with the transcription machinery to facilitate PIC formation.
Regulation of core promoter activation
Step 2
Step 2: Initiation by Pol II and ‘promotor escape’
- RNA Polymerase II transitions from being “paused” at the promoter to actually starting transcription (called promoter escape).
- Cofactors (e.g., Mediator, CBP/p300) and TFIIH activate RNA Polymerase II by opening the chromatin and phosphorylating the Pol II CTD.
- CBP/p300 coactivators, which contain lysine acetyltransferases, mediate histone acetylation to loosen chromatin and provide access to the transcription machinery.
Regulation of core promoter activation
Step 3
Step 3: Promoter-Proximal Pausing and Pause-Release
- After initiating transcription, RNA Polymerase II often pauses near the promoter (within 30–50 nucleotides). This pause is regulated by factors like DSIF and NELF.
- To continue transcription (productive elongation), cofactors (e.g., BRD4, P-TEFb) release Pol II from the pause.
- CBP/p300 coactivators and the mediator complex continue to facilitate chromatin remodeling, ensuring Pol II progresses efficiently.
A prototypical transcription factor
A prototypical transcription factor
- The DNA-binding domain recognizes specific DNA sequences at transcription factor binding sites (TFBS).
- Examples for DNA-Binding Domains are: Leucine Zipper & Zn finger domain
- The effector domain regulates transcription factor activity, such as by binding ligands.
- It can mediate protein-protein interactions through domains like the BTB domain.
- It may have enzymatic activities, such as chromatin modification (e.g., SET domain).
- These domains enable transcription factors to control gene expression by interacting with DNA, proteins, and chromatin
How would you determine whether a newly identified protein acts as a transcription factor?
Check for DNA-Binding Capability
- Use assays like Electrophoretic Mobility Shift Assay (EMSA) or ChIP-seq to confirm if the protein binds to specific DNA regions, a key characteristic of transcription factors.
Identify DNA-Binding Domains
- Analyze the protein sequence for known DNA-binding motifs (e.g., zinc fingers, leucine zippers), as transcription factors typically have specific domains for DNA interaction.
Functional Assays
- Perform reporter gene assays to test whether the protein can activate or repress transcription when bound to its target DNA.
