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.