control of transcription and chromatin Flashcards
lectures 1-4
What is the difference between the template and coding strands in transcription?
The template strand (3’-5’) is used by RNA polymerase to synthesize RNA, while the coding strand (5’-3’) has the same sequence as the mRNA (except T is replaced by U).
What are the key components involved in transcription?
RNA polymerase, ATP, UTP, CTP, GTP, and specific transcription factors like σ70 in prokaryotes.
What is the role of the σ70 factor in prokaryotic transcription?
The σ70 factor is part of the RNA polymerase holoenzyme and helps recognize the promoter regions in bacterial DNA to initiate transcription.
What are promoters in transcription?
Promoters are DNA sequences that initiate transcription, with key regions like the -35 and -10 sequences in prokaryotes or TATA boxes in eukaryotes.
What are the consensus sequences in prokaryotic promoters?
The consensus sequences are the -35 and -10 regions (e.g., TTGACA and TATAAAT) that help in the binding of RNA polymerase for transcription initiation.
What are the core promoter elements in eukaryotic transcription?
Core promoter elements in eukaryotes include the TATA box, initiator (Inr), motif ten element (MTE), and downstream promoter element (DPE).
What are CpG islands and their role in transcription?
CpG islands are regions with a high frequency of CG sequences in the promoter region of genes, where methylation of cytosines typically silences gene expression.
What are UAS and Enhancers in eukaryotic transcription?
UAS (Upstream Activating Sequences) and enhancers are regulatory regions where activators bind to increase gene transcription.
What are Silencers and URS in eukaryotic transcription?
Silencers and URS (Upstream Repressive Sequences) are DNA elements where repressors bind to decrease transcription.
What is the role of RNA polymerase I, II, and III in eukaryotic cells?
RNA polymerase I transcribes rRNA, RNA polymerase II transcribes mRNA, and RNA polymerase III transcribes tRNA and other small RNAs.
How does RNA polymerase II in eukaryotes differ from bacterial RNA polymerase?
RNA polymerase II requires general transcription factors (GTFs) to initiate transcription and lacks a sigma factor like bacterial RNA polymerase.
What are the General Transcription Factors (GTFs) in eukaryotic transcription?
GTFs are proteins like TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH that are involved in assembling the pre-initiation complex (PIC) at the promoter.
What is the role of TFIIH in transcription?
TFIIH has helicase activity to unwind DNA at the promoter, and its kinase activity phosphorylates the C-terminal domain (CTD) of RNA polymerase II.
How does the C-terminal domain (CTD) of RNA polymerase II function during transcription initiation?
The CTD is phosphorylated during transcription initiation and helps recruit additional factors needed for elongation and processing of the nascent mRNA.
What is the function of TFIID in transcription?
TFIID binds to the TATA box and recruits other GTFs and RNA polymerase II to form the pre-initiation complex.
What is promoter clearance in transcription initiation?
Promoter clearance occurs when RNA polymerase II starts transcribing the gene after the formation of the open complex and release of some general transcription factors.
What is the function of the helix-turn-helix structure in the TFIID complex?
The helix-turn-helix structure of the TATA binding protein (TBP) in TFIID helps it to recognize and bind to the TATA box, initiating transcription.
How can reporter genes be used to study promoters?
Reporter genes like GFP, luciferase, and LacZ can be linked to a promoter to measure gene expression, location, and response to signals.
What is the Pre-Initiation Complex (PIC)?
The PIC is a large multi-protein complex formed by RNA polymerase II and general transcription factors at the promoter, required for transcription initiation.
What are the major steps involved in transcription initiation by RNA polymerase II?
1) Formation of the pre-initiation complex (PIC),
2) DNA unwinding by TFIIH,
3) Phosphorylation of the CTD,
4) RNA polymerase II begins transcription and clears the promoter.
What does TFIIH contribute to in transcription?
TFIIH provides helicase activity to unwind the DNA and kinase activity to phosphorylate RNA polymerase II’s C-terminal domain (CTD), enabling transcription initiation.
What is the role of UAS/Enhancer elements in transcription?
UAS (Upstream Activator Sequence) and enhancer elements bind activators that stimulate high levels of transcription by interacting with the core promoter and transcription machinery. They can be located close to the core promoter (promoter proximal) or far from the transcription start site (distal).
What are some common types of UAS/Enhancer elements?
GC Box (GGGCGG) - Binds Sp1
Octamer (ATTTGCAT) - Binds Oct-1
**CAAT Box **(GGCCAATCT) - Binds NFY
**SRE **(TGACTCA) - Binds Serum Response Factor (SRF)
**HSE **(CTNGAATNTTCTAGA) - Binds Heat Shock Factor (HSF)
What is the function of activation domains in activators?
Activation domains in activators are regions that lack structural conservation and interact with components of the transcriptional machinery to increase transcription levels. They typically interact with TAFs, TFIID, TFIIB, and other transcription factors.
What are some examples of activation domains?
Acidic Patch - e.g., VP16
Glutamine-rich - e.g., SP1
Proline-rich - e.g., Jun
How do activators work to stimulate transcription?
**Promote binding of additional activators.
Stimulate complex assembly **by recruiting TFIID, TFIIB, and Mediator to the core promoter.
Release stalled RNA polymerase to initiate transcription.
Modulate chromatin structure to facilitate complex formation.
What is Mediator and what is its role in transcription?
Mediator is a large complex that bridges the interaction between activators and RNA polymerase II, aiding in the recruitment of RNA pol II to the promoter and enhancing the formation of the pre-initiation complex (PIC).
What are the components of the Pre-Initiation Complex (PIC)?
The PIC consists of general transcription factors (GTFs) like TFIID, TFIIB, TFIIF, TFIIE, TFIIH, and RNA polymerase II. Activators interact with these factors to assemble the PIC.
What is the function of the Mediator complex?
Mediator helps to bring activators and RNA polymerase II together, facilitating transcription initiation. It is crucial for recruiting RNA pol II to the core promoter.
What are some in vitro methods used to analyze activators?
DNA Footprinting
Electrophoretic Mobility Shift Assay (Gel Shift)
In vitro Transcription Assays to measure transcription activity.
What is Chromatin Immunoprecipitation (ChIP) used for?
ChIP is used to identify the binding sites of activators on DNA. The method involves immunoprecipitating the protein-DNA complex and analyzing it using PCR or sequencing.
How do activators modulate chromatin to facilitate transcription?
Activators can remodel chromatin to make it more accessible to the transcriptional machinery, aiding in the formation of the pre-initiation complex and facilitating transcription initiation.
What is the purpose of Reporter Assays in studying activators?
Reporter assays use reporter genes (like GFP or luciferase) to measure the level of transcriptional activity, helping to assess how activators influence gene expression.
How do activators interact with Mediator and TFIID to enhance transcription?
Activators interact with TAFs in TFIID and specific subunits of Mediator to recruit the transcriptional machinery, enhancing the formation of the pre-initiation complex and boosting transcription initiation.
How do activators influence RNA polymerase II activity?
Activators can help release stalled RNA polymerase II at the promoter, ensuring continued transcription elongation after initiation.
What are the primary mechanisms by which activators stimulate transcription?
Recruitment of transcription machinery.
Activation of chromatin remodeling.
Facilitation of RNA polymerase release from pausing.
Promoting the binding of additional activators.
What is the significance of “promoter proximal” enhancer elements?
Promoter proximal enhancer elements are located close to the core promoter and bind to activators that are constitutively active, helping regulate basal (low) transcription levels by interacting with the transcription machinery.
What are the functions of response elements in enhancer/UAS elements?
Response elements are enhancer sequences that bind transcription factors whose activity is regulated by specific stimuli. These include factors such as serum response factors (SRF) for growth factors and heat shock factors (HSF) for heat shock.
How do activators influence transcription levels under different conditions?
Activators interact with specific enhancer sequences, and their activity can be modulated by external signals, such as heat shock or growth factors, leading to an increase in transcription levels from basal to activated levels.
What is combinatorial control of transcription?
Combinatorial control refers to the concept that different types and combinations of enhancer sequences and activators dictate the timing, location, and level of gene transcription, allowing fine-tuned regulation in response to various signals.
What is the role of general transcription factors (GTFs) in transcription?
General transcription factors like TFIIB, TFIID, and TFIIH are essential for the assembly of the pre-initiation complex (PIC), and they interact with RNA polymerase II to facilitate the initiation of transcription.
How do activators work to increase the rate of PIC formation?
increasing TFIID binding, increasing TFIIB binding, increasing RNA pol II recruitment
How do activators influence the release of stalled RNA polymerase II?
Activators can release RNA polymerase II that is stalled after initiating transcription. This is crucial for genes that need rapid transcriptional activation, such as heat shock genes like hsp70, where RNA pol II stalls and requires activator-mediated release to continue.
What role does chromatin remodeling play in transcription?
Chromatin remodeling, facilitated by activators, involves changes to the structure of nucleosomes or histones that allow for better accessibility of the DNA to the transcription machinery, enabling efficient transcription initiation.
What is the purpose of Electrophoretic Mobility Shift Assays (Gel Shift)?
Gel shift assays measure the binding of proteins, such as activators, to DNA by analyzing the change in mobility of the DNA-protein complex in a gel under non-denaturing conditions. This technique identifies specific protein-DNA interactions.
How do activators enhance transcription initiation?
Activators enhance transcription initiation by facilitating the binding of transcription factors, including TFIID, TFIIB, and Mediator, to the core promoter. This accelerates the assembly of the pre-initiation complex and initiates transcription at higher levels.
How do activators work through a modular structure?
Eukaryotic activators are modular, meaning they contain distinct functional domains, such as DNA binding domains (e.g., zinc fingers, leucine zippers) and activation domains (e.g., acidic patches), which work together to regulate gene expression effectively.
What is chromatin?
Chromatin is the protein-DNA complex that packages eukaryotic DNA into a compact form, enabling it to fit within the cell nucleus.
Why is DNA packaging necessary in eukaryotic cells?
The human genome is approximately 2 metres long and must be compacted to fit into a nucleus just a few microns in diameter.
What is the primary function of chromatin?
To compact DNA and regulate access to the genetic information.
What proteins primarily compose chromatin?
Chromatin is composed mainly of small, basic proteins called histones.
What are the two main types of histones?
Core histones (H2A, H2B, H3, H4) and linker histones (e.g., histone H1).
What is the nucleosome?
The nucleosome is the fundamental repeating subunit of chromatin, consisting of ~147 base pairs of DNA wrapped twice around a histone octamer.
What is the composition of the histone octamer?
central H3-H4 tetramer and two flanking H2A-H2B dimers.
What structural change occurs when linker histones bind DNA?
Linker histones help form a thicker 30 nm fibre from the 10 nm “beads-on-a-string” structure in vitro.
What are histone variants?
Histone variants are alternative forms of histones encoded by distinct genes, which confer novel structural and functional properties to nucleosomes.
Which conventional histones have variants?
All conventional histones except H4 have known variants, such as H2A.Z, H2AX, and CENP-A.
What are post-translational modifications of histones?
Modifications include acetylation, methylation, ubiquitylation, and phosphorylation, which occur on histone N-terminal tails.
How can histone modifications influence transcription?
They can alter chromatin structure or recruit non-histone proteins that affect transcriptional machinery.
What enzymes control histone acetylation?
Histone acetyltransferases (HATs) mediate acetylation, while histone deacetylases (HDACs) reverse it.
How does acetylation of histones affect transcription?
Acetylation correlates with transcriptional activation by loosening chromatin structure and recruiting bromodomain proteins.
What is the “histone code”?
The hypothesis that specific combinations of histone modifications determine chromatin’s transcriptional state.
What are bromodomains?
Protein domains that recognize acetylated lysine residues and often promote transcription.
What enzymes mediate histone methylation?
Histone lysine methyltransferases (HKMTs), often containing a SET domain.
What is the functional significance of histone methylation?
Depending on context, methylation can either activate or repress transcription
What are examples of histone methylation marks and their effects?
H3 Lys4: Activation
H3 Lys27: Repression
H3 Lys36: Activation
How does chromatin inhibit transcription?
Chromatin compacts DNA, blocking access to transcription machinery.
What experimental evidence shows chromatin blocks transcription?
In vitro, transcription occurs on naked DNA but not on chromatin templates without additional modifications.
What happens to nucleosomes during transcriptional activation?
Nucleosomes are disrupted or lost to allow access to the transcription machinery.
What are the three mechanisms that modulate chromatin structure?
- Histone variants
- Post-translational modifications
- ATP-dependent chromatin remodelling.
What is ATP-dependent chromatin remodelling?
It uses ATP to reposition or evict nucleosomes, altering chromatin accessibility.
How do activators influence chromatin?
Activators recruit HATs and other modifying enzymes to specific promoters, enabling transcription.
What is the role of HAT complexes in transcription?
HAT complexes acetylate histones, promoting chromatin relaxation and transcription activation.
What is the GAL4 promoter system?
A yeast model where the GAL4 promoter is active in galactose but repressed in glucose, allowing controlled histone depletion experiments.
What happens to gene expression when nucleosomes are depleted?
Inducible genes are more readily expressed due to increased access for transcription machinery.
What are some examples of HAT families?
GNAT family: Yeast SAGA, Human STAGA
MYST family: Yeast NuA4, Human Tip60.
What is the relationship between chromatin structure and transcription?
Chromatin dynamically balances DNA compaction with accessibility for transcription, modulated by histone modifications and chromatin remodelling.
What is the “beads-on-a-string” chromatin structure?
It refers to the 10 nm fibre where nucleosomes appear as discrete “beads” connected by linker DNA.
What is the proposed 30 nm fibre in chromatin organisation?
A more compact chromatin fibre formed with linker histones, though evidence suggests this structure is unlikely to exist in vivo.
What subunits in HAT complexes interact with transcriptional activators?
Examples include Tra1 in yeast and TRRAP in mammals, which link HATs to specific promoters.
How do bromodomain proteins influence transcription?
Bromodomain proteins bind acetylated lysines, recruiting transcription machinery to promote gene expression.
How does histone methylation differ from acetylation?
Methylation is less readily reversible and does not affect charge but serves as a binding signal for specific proteins.
What are the conflicting roles of nucleosomes in the nucleus?
Nucleosomes compact DNA but also serve as a template for transcription, necessitating dynamic regulation.
How are nucleosomes disrupted during transcription?
Nucleosomes are repositioned or evicted to allow access to the transcriptional machinery.
What is the significance of nucleosome positioning experiments?
These experiments showed that nucleosomes are disrupted or lost during transcriptional activation in vivo.
What did the GAL4 promoter experiment in yeast demonstrate?
It showed that nucleosome depletion via histone H4 suppression increases the expression of inducible genes.
What is ATP-dependent chromatin remodelling?
It is the process of using ATP to reposition or evict nucleosomes, altering chromatin accessibility for transcription.
Can you name some ATP-dependent chromatin remodelling complexes?
Examples include SWI/SNF, ISWI, and CHD complexes.
What is the “histone fold domain”?
A structural motif in core histones enabling the “handshake” interaction to form the histone octamer.
What is the role of post-translational modifications in chromatin dynamics?
They serve as recruitment signals for proteins like bromodomains or chromodomains, influencing chromatin structure and transcription.
What are chromodomain-containing proteins?
Proteins that recognise methylated lysine residues on histones, often influencing transcriptional repression or activation.
What are the major mechanisms for modulating chromatin structure and function?
Post-translational modifications of histones (acetylation, methylation, ubiquitylation, phosphorylation), ATP-dependent chromatin remodelling, and chromatin-associated proteins.
What is the “Histone Code”?
A hypothesis suggesting that specific combinations of histone post-translational modifications act as a code to regulate chromatin structure and transcription.
What enzymes mediate histone acetylation?
Histone acetyltransferases (HATs).
What enzymes mediate histone deacetylation?
Histone deacetylases (HDACs).
What is the role of histone methylation?
It can either activate or repress transcription depending on the site of methylation (e.g., H3K4me activates, while H3K9me represses).
Which enzymes catalyse histone methylation?
Histone methyltransferases.
Which enzymes remove histone methylation marks?
Histone demethylases.
What does histone ubiquitylation involve?
The addition of ubiquitin molecules to histones, often regulating DNA repair and transcription.
What is histone phosphorylation?
The addition of phosphate groups, often linked to chromatin condensation and DNA damage responses.
What are bromodomain proteins?
Proteins that recognise and bind acetylated lysines on histones.
What are chromodomain proteins?
Proteins that recognise and bind methylated lysines, such as H3K9me.
What are PWWP and PhD proteins?
Domains found in proteins that bind specific histone modifications, aiding in chromatin organisation and function.
What is ATP-dependent chromatin remodelling?
The use of ATP to slide, unwrap, evict nucleosomes, or exchange histone variants to regulate chromatin accessibility.
What families of ATP-dependent remodellers exist?
SWI/SNF, ISWI, CHD, and INO80 complexes.
What is the Snf2 ATPase?
A catalytic subunit in SWI/SNF complexes that hydrolyses ATP to remodel nucleosomes.
How does SWI/SNF remodel chromatin?
By inducing torsion through ATP hydrolysis, disrupting histone-DNA interactions, and moving nucleosomes.
How do ATP-dependent and HAT complexes cooperate?
HATs acetylate histones to recruit ATP-dependent remodellers like SWI/SNF via bromodomain interactions.
What is the role of SWI/SNF in yeast?
It regulates transcription of ~5% of genes and plays a role in cell cycle regulation.
How is SWI/SNF linked to cancer in humans?
Mutations in SWI/SNF subunit genes are found in 25% of cancers, impairing tumour suppressor pathways.
What are the three related SWI/SNF complexes in humans?
cBAF, PBAF, and ncBAF.
What is the role of HDACs in transcriptional repression?
They remove acetyl groups, causing chromatin compaction and repressing transcription.
What is the NuRD complex?
A transcriptional repressor that combines HDAC activity with ATP-dependent remodelling.
What are key characteristics of heterochromatin?
Hypoacetylation, specific histone methylation (H3K9me, H3K27me), and transcriptional silencing.
What regions are commonly heterochromatic?
Centromeres, telomeres, and the inactivated X chromosome.
What is the role of HP1 in heterochromatin?
HP1 binds H3K9me, compacts chromatin, and recruits silencing factors.
How can reporter silencing assays study heterochromatin?
By measuring gene expression changes in heterochromatic versus euchromatic regions.
What controls X-chromosome inactivation?
The non-coding RNAs Xist and Tsix, which regulate the formation of a Barr body.
What is the role of Xist in X-chromosome inactivation?
Xist coats the inactive X chromosome and recruits silencing factors.
What distinguishes euchromatin from heterochromatin?
Euchromatin is gene-rich and transcriptionally active, while heterochromatin is gene-poor and silenced.
What is a common feature of all ATP-dependent remodellers?
A Snf2-related ATPase domain that uses ATP to remodel chromatin.
How is SWI/SNF involved in development?
It regulates key genes; deletion in mice causes embryonic lethality.
What is the function of histone lysine methylases in heterochromatin formation?
They catalyse the methylation of H3K9, which recruits HP1 and silencing factors.
What are Class I, II, III, and IV HDACs?
Classes of HDACs differing in co-factors and roles, including SIRTUINS requiring NAD+.
How do transcription factors recruit chromatin remodellers?
By binding to specific domains, such as bromodomains or chromodomains.
What did cryo-EM studies of SWI/SNF reveal?
Structural insights into its interaction with nucleosomes, showing how remodelling occurs.
