lecture 3 Flashcards
how is transcription regulated by activators?
- promotes binding of additional activator
- stimulates complex assembly
- release stalled RNA polymerase II
what is the basic function of chromatin?
to compact dna
what is the composition of chromatin?
-primarily composed of small basic proteins called histones
what are the two types of histones?
core and linker
what are the properties of core histones?
-N terminal tail (rich in lysine and arginine)
-globular domain (alpha helices and loops)
-H2A, H2B, H3, H4
how do core histones bind DNA?
-form repeating units called nucleosomes
-only stable when DNA is wrapped around
what is a nucleosome?
~147 bp of DNA wrapped twice around an octamer of histone proteins
how are nucleosomes organised?
- DNA passes directly from one nucleosome to the next
- linker histones (histone 1) bind to DNA between nucleosomes
- in vitro linker histones result in formation of thicker 30nm fibre
what is the evidence that chromatin inhibits transcription?
- in vitro reconstitution experiments
- in vivo nucleosome positioning experiments (nucleosomes are duplicated/lost during transcriptional activation)
- genetic studies in S. cerevisiae
what occurs in the in vitro reconstitution experiments?
RNA pol II + txn factors + naked DNA template = transcription
RNA pol II + txn factors + chromatin template = no transcription
what is shown from the S.cerevisae genetic studies?
-nucleosome number was experimentally controlled
-chromosomal copies of H4 genes were deleted and plasmid expressing H$ is present (GAL4 promoter)
what happens when galactose is present?
the GAL4 promoter is switched on
what happens when glucose is present?
GAL4 promoter is OFF, H4 expression rapidly shut off, causes nucleosome depletion
why is chromatin structure dynamic?
can be regulated and modified to allow transcription and regulate gene expression
what 3 mechanisms modulate the structure of chromatin?
-histone variants
-post translational modification of histones
-ATP dependent chromatin remodelling
what are histone variants?
-encoded by genes that differ from highly conserved major types
what are the post translational modifications of histones?
acetylation. methylation, ubiquitylation, phosphorylation
help to control gene expression
how can histone modification effect the transcriptional state?
-could directly alter chromatin folding/structure by modifying tails
-could control recruitment of non histone proteins to chromatin (influences recruitment of transcriptional machinery)
what is histone lysine acetylation mediated by?
HATs, is readily reversible by HDACs
highly dynamic acetylation
what proved that acetylation is a key component of transcriptional activation?
Gcn5 is a transcriptional activator
how are HATs recruited?
-activators recruit HATs to specific promoters
-once bound, acetylates nucleosomes in close vascinity
-activator interacts with the UAS and Tra1 acts directly with the activator once bound
how does acetylation mediate transcriptional activation (1)?
- direct influence on chromatin structure
-lysine acetylation reduces the positive charge
-positively charged lysine in flexible tails can make interactions with negatively charged DNA in nucleosomes
-compacts the chromatin structure
-high acetylation = reduced potential for interaction
how does acetylation mediate transcriptional activation (2)?
- directs the recruitment of bromodomain proteins
-specific acetylated lysine residues are recognised by proteins with bromodomains
-bromodomains often promote transcription
where does histone methylation occur?
on lysine and arginine (less understood) residues
