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Questions and Answers
What is the primary function of enhancers in mammalian genomes?
What is the primary function of enhancers in mammalian genomes?
Which of the following statements accurately describes silencers?
Which of the following statements accurately describes silencers?
What is the role of insulators in DNA?
What is the role of insulators in DNA?
What must occur for RNA polymerase to initiate transcription at eukaryotic promoters?
What must occur for RNA polymerase to initiate transcription at eukaryotic promoters?
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What is the typical length of a locus control region (LCR)?
What is the typical length of a locus control region (LCR)?
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What is the initial event that occurs during the multistage process of transcription initiation?
What is the initial event that occurs during the multistage process of transcription initiation?
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What is the role of Positive transcription elongation factor-b (P-TEFb) during transcription?
What is the role of Positive transcription elongation factor-b (P-TEFb) during transcription?
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How does the transition to productive elongation in transcription occur?
How does the transition to productive elongation in transcription occur?
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What occurs during promotor escape in the transcription process?
What occurs during promotor escape in the transcription process?
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Which factor is crucial for determining whether a poised or active gene is transcribed?
Which factor is crucial for determining whether a poised or active gene is transcribed?
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What is one of the main functions of TFIIH during transcription initiation?
What is one of the main functions of TFIIH during transcription initiation?
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How does the Mediator complex assist in the transcription process?
How does the Mediator complex assist in the transcription process?
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Which activity is NOT associated with TFIIH?
Which activity is NOT associated with TFIIH?
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What is the approximate number of proteins that make up the Mediator complex?
What is the approximate number of proteins that make up the Mediator complex?
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What is a crucial role of TFIIE in the initiation of transcription?
What is a crucial role of TFIIE in the initiation of transcription?
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Which RNA polymerase is primarily associated with the functions of TFIIH?
Which RNA polymerase is primarily associated with the functions of TFIIH?
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What does the CTD tail of RNA polymerase II interact with in the Mediator complex?
What does the CTD tail of RNA polymerase II interact with in the Mediator complex?
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Which activity of TFIIH is crucial for separating the DNA strands at the transcription start site?
Which activity of TFIIH is crucial for separating the DNA strands at the transcription start site?
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What is indicated by the presence of Topologically Associated Domains (TADs) in multicellular eukaryotes?
What is indicated by the presence of Topologically Associated Domains (TADs) in multicellular eukaryotes?
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How do transcriptionally active genes relate to nuclear pore complexes?
How do transcriptionally active genes relate to nuclear pore complexes?
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What is a consequence of disrupted nuclear membrane in progeria patients?
What is a consequence of disrupted nuclear membrane in progeria patients?
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What role does the nuclear lamina play in eukaryotic cells?
What role does the nuclear lamina play in eukaryotic cells?
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What effect does the protein SIRT6 have in relation to premature aging syndromes?
What effect does the protein SIRT6 have in relation to premature aging syndromes?
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What changes occur to chromosome territories during transcriptional activity?
What changes occur to chromosome territories during transcriptional activity?
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How does the spatial organization of the nucleus relate to cellular health?
How does the spatial organization of the nucleus relate to cellular health?
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Which of the following is NOT a function of the nuclear lamina?
Which of the following is NOT a function of the nuclear lamina?
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What is the role of nuclear localization signal (NLS) in proteins targeted to the nucleus?
What is the role of nuclear localization signal (NLS) in proteins targeted to the nucleus?
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Which of the following proteins is primarily involved in the nuclear export process?
Which of the following proteins is primarily involved in the nuclear export process?
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What is the function of the poly(A) tail in mRNA?
What is the function of the poly(A) tail in mRNA?
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Which factor is NOT involved in the cleavage reaction to generate a 3′ end of mRNA?
Which factor is NOT involved in the cleavage reaction to generate a 3′ end of mRNA?
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What sequence signals for the cleavage during the polyadenylation of mRNA?
What sequence signals for the cleavage during the polyadenylation of mRNA?
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Which element is associated with the site of cleavage for polyadenylation?
Which element is associated with the site of cleavage for polyadenylation?
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What type of diffusion occurs through nuclear pore complexes (NPCs) for small molecules?
What type of diffusion occurs through nuclear pore complexes (NPCs) for small molecules?
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What do importins and exportins belong to?
What do importins and exportins belong to?
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What is the role of promoter clearance in transcription?
What is the role of promoter clearance in transcription?
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Which phosphorylation on the CTD is required for the transition to elongation?
Which phosphorylation on the CTD is required for the transition to elongation?
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Which of the following describes the role of CTD phosphorylation at Ser2?
Which of the following describes the role of CTD phosphorylation at Ser2?
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What is a function of the Paf1 complex?
What is a function of the Paf1 complex?
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How does the CTD affect RNA processing and export?
How does the CTD affect RNA processing and export?
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What happens to RNA polymerase during the initiation phase?
What happens to RNA polymerase during the initiation phase?
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What determines whether a gene will remain in a poised state or become active?
What determines whether a gene will remain in a poised state or become active?
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What is the main requirement for the capping of mRNA in transcription?
What is the main requirement for the capping of mRNA in transcription?
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During the transcription process, what is the function of the RNA polymerase's CTD?
During the transcription process, what is the function of the RNA polymerase's CTD?
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What effect does enhancing phosphorylation levels of the CTD tail have on transcription?
What effect does enhancing phosphorylation levels of the CTD tail have on transcription?
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Study Notes
Eukaryotic Gene Expression: Transcription
- Eukaryotic transcription takes place on a densely proteinized chromatin template.
- Bacterial RNA polymerase reads DNA; eukaryotic RNA polymerase cannot directly read DNA.
- Transcription requires sequence-specific DNA-binding transcription factors (TFs).
- Core RNA polymerase II machinery is essential.
- Coregulators bridge TFs to the transcriptional machinery.
- Chromatin remodeling factors mobilize nucleosomes.
- Enzymes catalyze covalent modifications of histones and other proteins.
- Eukaryotic transcription is usually under positive regulation.
- Opening chromatin and binding TFs to the binding sites precede polymerase binding.
- Polymerase binds to promoter and TFs bind to enhancers.
Eukaryotic Transcriptional Regulation
- Eukaryotic genomes fold into topologically associated domains (TADs).
- Chromosomes with low gene density reside at the nuclear periphery.
- Chromosomes with high gene density occupy the nuclear interior.
- Transcriptionally active genes associate with nuclear pore complexes.
- Transcription decondenses chromosome territories, loops back into condensed territories when transcription ceases.
- The banding pattern of polytene chromosomes corresponds to TADs.
Nuclear Architecture and Premature Aging Syndromes
- A link exists between disrupted nuclear membrane and premature aging syndromes.
- Nuclear lamina: a protein meshwork underlying the nuclear membrane composed of intermediate filaments.
- Nuclear lamina provides mechanical support, regulates cellular events (DNA replication), and participates in chromatin organization.
- It anchors nuclear pore complexes and is a platform for protein complexes involved in signal transduction pathways.
Eukaryotic Gene Expression: Transcription
- Some eukaryotic TFs function by introducing DNA bends, facilitating interactions of other components.
Nucleic Acid Recognition by Proteins
- DNA-binding proteins incorporate domains that facilitate the binding of nucleic acid.
- These proteins interact with DNA ends (e.g., DNA ligases, exonucleases), enclose DNA, bind to the face of DNA's helix, and possess motifs that fit into the major grove of DNA for higher stability.
Long Range Regulatory Elements
- Enhancers: typical mammalian genomes contain many enhancers (1-2% contribute to gene regulation); ranging from 100-500 bp in length; can increase or decrease the promoter.
- Silencers: decrease gene promotor activity; between 700 and 1000 bp.
Initiation in Eukaryotic Promoters
- Various basal transcription factors (BTFs) bind to cis-acting elements to form the initiation complex for RNA polymerase.
- Basal factors are identified as TFIIA, TFB, TFIIIC, TFIIID, etc.
- Core promoter motifs for RNA polymerase II are present.
Eukaryotic RNA Polymerases and Promoter Recognition
- Eukaryotes have multiple nuclear DNA-dependent RNA polymerases (I, II, III).
- Different RNA polymerases transcribe different products within the cell compartment (e.g., rRNA, mRNA, tRNA).
RNA Polymerases II Promoters
- Enhancers and silencers increase or decrease the expression of a gene.
- Enhancers function upstream (or downstream) of the promoter.
- Enhancers and silencers may be separated from the promoter by distances as large as 100 kb.
Enhancers Assist Initiation
- Enhancers activate an accessible promoter located virtually anywhere from the promoter either upstream or downstream.
- Enhancers increase activator concentration.
How Enhancers Work
- Enhancers normally work in cis configuration, with a target promoter; enhancers can be made to work in trans configuration;
- The principle is that enhancers work in any situation and distance from the promoter.
- Enhancers increase activator concentration in the vicinity of the promoter from cis.
Role of Coactivators and Corepressors
- Coactivators increase or corepressors decrease transcriptional activity.
- Coactivators can be chromatin modification complexes (histone modifications) or chromatin remodeling complexes.
Eukaryotic RNA Polymerases
- Typical eukaryotic RNA polymerases have 12 subunits (2 large, many smaller).
- RNA polymerase II is the most characterized.
- The largest subunit in RNA polymerase II has a carboxy-terminal domain (CTD).
Phosphorylation of the RNA Polymerase II C-Terminal Doamin (CTD)
- CTD has a repeating sequence of seven residues (Y1S2P3T4S5P6S7); the number of repeats can differ among species.
- Phosphorylation status affects mRNA processing stage transitions
Mitochondrial and Chloroplast RNA Polymerases
- Mitochondrial and chloroplast RNA polymerases are smaller than bacterial enzymes.
- Eukaryotic genomes have fewer genes to transcribe and simpler regulation.
RNA Polymerase I
- RNA polymerase I directs RNA synthesis in the nucleoli and transcribes ribosomal RNA.
RNA Polymerase III Promoter Organization
- RNA polymerase III uses two classes of promoters: internal promoters and external promoters.
- Specific promoter elements and their positioning are different for internal versus external promoters.
Interactions at Internal Promoters
- Protein-protein interactions assist in the assembly of polymerase with basal factors.
- TAFs and TFB bind to different regions of the promoter.
Interactions at External Promotors
- TATA boxes, proximal sequence elements (PSEs), and octamer elements aid in basal transcription and efficiency.
Chromatin Remodeling Exposes the Promoter
- Chromatin remodeling occurs before the initiation of transcription, as nucleosomes must be moved away from the promoter to expose it to transcription factors.
TATA Binding Proteins (TBP): A Nearly Universal Factor
- TBP is a component of the positioning factor (interaction with RNA polymerase I/II/III) required for promoter binding.
TATA-Less Pre-initiation Complex
- TATA-less promoters rely on other elements for positioning and can be found in housekeeping and developmental genes.
- TBP has similar positioning in TATA-less promoters.
Gene Promoters and Chromatin Categories
- The inaccessibility of DNA due to histone proteins correlates to various chromatin categories.
- Poised genes require secondary signals while active genes only require basal TFs.
Transcription Basal Apparatus Assembles at the Promoter
- TFIID is the first general TF to associate with template DNA.
- Factors such as activators, UPEs and UPEs' binding factors increase the frequency of initiation.
- Binding TFIID to TATA box or Inr triggers the first step of initiation in transcription.
Transcription Basal Apparatus Assembles at the Promoter
- TFIIH is a complex TF and is involved in DNA repair.
- TFIIH phosphorylates CTD tail of RNA pol II, which is involved in release for further progress to elongation.
Mediator: A Molecular Drawbridge
- Mediator connects transcriptional activators (bound at enhancers and other regulatory elements) to RNA polymerase II
- The mediator links the initiation complex with distal elements by acting as a drawbridge.
Initiation of Transcription
- Multistage process: includes promoter melting, abortive initiation (short transcripts), promoter escape, pausing, and pause release.
Promoter Clearance and Elongation
- DNA strands separate following RNA pol binding.
- Promoter clearance is a crucial determinant for poised or active gene transcription.
- Enhancers influence the process.
The C-terminal Domain (CTD) of RNA Pol II
- CTD is required for capping and splicing; a repetitive structure emerged via evolution.
- CTD contains repeats (7 amino acids), linking transcriptional and RNA processing.
- Phosphorylation of Serines in the CTD is crucial for releasing the RNA Pol II for elongation.
Co-transcriptional Processing
- Transcription and co-transcriptional processing are tightly linked and performed via multifunctional proteins (e.g., capping enzyme complex (CEC)).
- The processing occurs concurrently with transcription, so RNA processing begins immediately after transcription, using 5' capping as an initial example.
The Cap-Binding Complex
- Cap binds to the cap-binding complex (CBC), which is identified by the nuclear pore.
- This complex facilitates mRNA export and prevents decapping enzymes.
- CBC is exchanged with other factors during mRNA transport and translation.
Nuclear Import and Export
- Small and large molecules are actively transported across the nuclear membrane.
- Importins and exportins (karyopherins) are the receptors involved in this process, and Ran is a vital factor for these processes.
The Ran Cycle
- Ran is a critical factor for nuclear transport and regulates processes within the import/export of molecules into and out of the nucleus.
Regulated Nuclear Import and Export of TFs and Proteins
- Regulatory proteins need to be directed to their corresponding nuclear activity sites.
- The receptors are known as importins and exportins (karyopherins) and mediate nuclear import and export pathways.
Nuclear Import and Export: TFs, Proteins
- Mechanisms in nuclear import/export: importin/Ran-dependent processes and nuclear import, and nuclear export.
Termination of Transcription
- The process is initiated by recognition of the sequence AAUAAA in Poly(A) sites.
- The endonuclease cleaves RNA, and Poly(A) polymerase adds 200 A residues to the 3' end.
Cleavage and Poly(A) at the 3' end
- CPSF/CstF and PAP proteins are part of the 3' processing complex for mRNA cleavage and polyadenylation as well as degradation avoidance.
Histone mRNA 3ʹ End Formation
- Histone mRNAs lack introns and contain HDE sequences at their 3ʹ ends, which are recognized by factors and regulate their localization.
- These elements are vital for mRNA regulation.
Summary: Initiation is Followed by Promoter Clearance and Elongation
- RNA pol II promoters' key regulated steps are promoter clearance and elongation, controlled via enhancers and phosphorylation of the CTD tail.
- These events involve TFs and accessory factors.
Other Core Promotor Elements
- Additional elements (e.g, BRE, Inr, MTE, DPE) help regulate transcription initiation in promoters lacking TATA box; TATA box, and upstream promoter elements (UPEs) are present.
Transcription Elongation Through the Nucleosome Barrier
- Nucleosomes serve as barriers to elongating RNA polymerases II.
- Chromatin remodeling complexes (e.g., FACT, elongation factors, and TFIIS) facilitate the movement of the RNA polymerase over nucleosomes.
Co-transcriptional Processing Revisited
- Transcription and processing are coupled, utilizing various factors to ensure timely and accurate transcript production and modification (e.g., capping, splicing).
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Description
This quiz covers key concepts related to transcription regulation in mammalian genomes, including the roles of enhancers, silencers, insulators, and various transcription factors. Test your understanding of the transcription initiation process and the functions of different complexes involved. Ideal for students studying molecular biology and genetics.
