Lecture 4: Flashcards
What are the 3 RNA polymerase types in eukaryotes and what do they do?
RNA Pol I: involved in making ribosomal RNA
RNAPol II: transcribed all messenger RNAs, protein coding genes, and non-coding genes
RNA Pol III: make different types of non-coding RNA
What must all RNA polymerases have?
Ways of getting DNA through, bringing in ribonucleotides, and have a channel for RNA to exit
What roles does GTFII have?
- binds at the core promoter and positions Pol II where transcription will start
- aids in pulling apart 2 strands
- helps release RNA Pol II to start elongation
What roles does GTFII have?
- binds at the core promoter and positions Pol II where transcription will start
- aids in pulling apart 2 strands
- helps release RNA Pol II to start elongation
What does TATA binding protein do?
Binds the TATA sequence in the minor groove of DNA, inducing an 80 degree kink
What steps and transcription factors are involved in Pol II transcriptional initiation?
TFIID: binds TATA sequence
TFIIB: recignises BRE element, helps with positioning
TFIIF: stabilizes interactions between Pol II and other factors, helps attract TFIIE & TFIIH
TFIIE: attracts and regulates TFIIH
TGIIH: unwinds DNA and phosphorylates Pol II tail
What is TFIIH?
DNA helicase that hydrolyses ATP and unwinds DNA
Phosphorylates Ser5 in the CTD tail which shifts period of abortive initiation to elongation
What is the role of general transcription factors?
Do decide where RNA pol II is going to start transcribing
What are activators and repressors and what do they do?
Are protein that interact directly with DNA upstream of the promoter, and define when and how much of a gene gets produced
How do activators make DNA accessible?
Bind to specific elements of DNA upstream of promoter and recruits acetylases
What is the purpose of basal apparatus?
Determines start point for transcription
In what ways can co-activators change chromatin structure?
Covenant histone modifications, nucleosome remodelling, nucleosome removal, and histone replacement
In what ways do different factors effect transcription?
- RNA polymerase and basal factors bind at promoter
- activators bind at promoter
- activators bind to distal sites in promoter or to enhancers
- co-activators connect activators to basal factors
- co-activators or regulators act on local structure of the gene
In what ways can repressors block activators?
- competitive DNA binding
- masking activation surface
- direct interaction with GTFs
What molecules can repressors recruit?
- chromatin remodelling complexes
- histone deacetylases
- histone methyltransferases
What do mediators do?
Bonds Pol II and its initiation factors and can enhance recruitment of Pol II to activator bound DNA
How does induction of IFNI during viral infection initiate transcription?
- infection triggers multiple different activators
- activate IFN-B which is then secreted by infected cells
- secretion warns uninfected cells into an antiviral state
- activators cooperatively bind to sites within enhancer upstream from promoter
What’s the theory behind cooperative binding?
- individual activators bind DNA weakly on their own
- together they bind cooperatively
- once bound they recruit the general machinery to generate transcription
What method is used to determine what regulatory protein bind in vivo?
ChIP
What method is used to determine what regions of DNA are bound to proteins in vitro?
Foot printing/ EMSA
What is the underlying concept of DNA foot printing assay?
DNA that is bound to protein is protected from chemical and enzymatic degradation
What are the steps involved with DNA footprinting experiments?
- synthesise or amplify DNA of interest
- label it radioactively or with fluorescence
- incubate with protein
- cleave it using agent such as DNase or hydroxyradical
- visualise resulting pattern next to sequencing ladder
What is the theory behind electrophoretic mobility shift assay?
Protein binding will induce a change in mobility of DNA on a gel
What is needed for EMSA?
- purified protein or lysate
- good antibody to the protein
- synthetic DNA and labelling option
