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Molecular Genetics > Lecture 4: > Flashcards

Lecture 4: Flashcards

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1
Q

What are the 3 RNA polymerase types in eukaryotes and what do they do?

A

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

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2
Q

What must all RNA polymerases have?

A

Ways of getting DNA through, bringing in ribonucleotides, and have a channel for RNA to exit

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3
Q

What roles does GTFII have?

A
  • 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
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4
Q

What roles does GTFII have?

A
  • 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
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5
Q

What does TATA binding protein do?

A

Binds the TATA sequence in the minor groove of DNA, inducing an 80 degree kink

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6
Q

What steps and transcription factors are involved in Pol II transcriptional initiation?

A

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

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7
Q

What is TFIIH?

A

DNA helicase that hydrolyses ATP and unwinds DNA

Phosphorylates Ser5 in the CTD tail which shifts period of abortive initiation to elongation

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8
Q

What is the role of general transcription factors?

A

Do decide where RNA pol II is going to start transcribing

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9
Q

What are activators and repressors and what do they do?

A

Are protein that interact directly with DNA upstream of the promoter, and define when and how much of a gene gets produced

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10
Q

How do activators make DNA accessible?

A

Bind to specific elements of DNA upstream of promoter and recruits acetylases

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11
Q

What is the purpose of basal apparatus?

A

Determines start point for transcription

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12
Q

In what ways can co-activators change chromatin structure?

A

Covenant histone modifications, nucleosome remodelling, nucleosome removal, and histone replacement

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13
Q

In what ways do different factors effect transcription?

A
  • 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
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14
Q

In what ways can repressors block activators?

A
  • competitive DNA binding
  • masking activation surface
  • direct interaction with GTFs
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15
Q

What molecules can repressors recruit?

A
  • chromatin remodelling complexes
  • histone deacetylases
  • histone methyltransferases
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16
Q

What do mediators do?

A

Bonds Pol II and its initiation factors and can enhance recruitment of Pol II to activator bound DNA

17
Q

How does induction of IFNI during viral infection initiate transcription?

A
  • 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
18
Q

What’s the theory behind cooperative binding?

A
  • individual activators bind DNA weakly on their own
  • together they bind cooperatively
  • once bound they recruit the general machinery to generate transcription
19
Q

What method is used to determine what regulatory protein bind in vivo?

A

ChIP

20
Q

What method is used to determine what regions of DNA are bound to proteins in vitro?

A

Foot printing/ EMSA

21
Q

What is the underlying concept of DNA foot printing assay?

A

DNA that is bound to protein is protected from chemical and enzymatic degradation

22
Q

What are the steps involved with DNA footprinting experiments?

A
  • 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
23
Q

What is the theory behind electrophoretic mobility shift assay?

A

Protein binding will induce a change in mobility of DNA on a gel

24
Q

What is needed for EMSA?

A
  • purified protein or lysate
  • good antibody to the protein
  • synthetic DNA and labelling option
25
Q

What is ChIP used for?

A

Determines all regulatory sequences occupied by given transcription regulator, pulls out protein and identifies what its associated with

26
Q

What steps are taken in ChIP experiments?

A
  • cross link proteins to DNA with formaldehyde
  • lyse cells and break DNA into small fragments
  • precipitate DNA using antibodies against gene regulatory protein A
  • reverse formaldehyde cross links and remove protein
  • amplify precipitated DNA using PCR

Molecular Genetics (20 decks)

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