Gene-L13-Transcription 5: Regulation of Transcription

how is gene expression regulated at the level of transcription INITIATION?

• genes are expressed at different efficiencies- even when using the same RNA polymerase and core promtor elements

• regulation determines is transcription is at high levels or low levels

• more stable transcription complexes- mor etranscription

• less stable- reduces transcription

how do enahancers and regulatory proteins influence basal transcription machinery?

• regulatory proteins bind enhancers which can be activators, repressors or weak modulators that fine tune

• enhancers can act far from the TATA box- and influence transcription

• regulate basal transcription- General transcription factors and RNA pol II

MECHANISM:

• DNA looping brings enhancer bound proteins to the promotor- stabilise or destabilise the pre-initiation complex

• stronger enhancer activity→ more transcription initiation

• repressors→reduces initiation probability

how do transcription activators work? mechanise 4

• one activator binds DNA and then recruits more regulatory proteins

• activators interact with RNA pol II and general Tfs

• activators help release th pol II when it pauses after 30 nucleotides

• promotes stable and active transcription complex and increases the likelihood off productive RNA synthesis

how do eukaryotic transcription repressors work? mechanism

1. competitive DNA binding- binds to the same or overlapping DNA sequence as an activator does. blocks transcription initiation by activator- regulated by signalling

2. protein-protein inhibition- repressors can bind directly to an activator protein and mask/block the activation domain- no transcription machinery

3. direct inhibition of GTFs- repressor interacts with the basal transcription machinery. stops proper assembly/function of the pre-initiation complex

why are the recruitment of cofactors necessary? when are they necessary? evidence of this? mechanism

• activators generally don’t act directly on RNA pol II or GTFs alone- need mediator complexes to transmit effect

• experiment: GTF+nucleotides- low basal transcription

• activator alone→ little increase

• activator and cell lysate co factors→ strong increase

mechanism:

• activators have 2 domains- DNA binding domain such as zinc fingers and activation domains which recruits regulatory machinery

• activation recruits cofactors- not always GTFs

• cofactors act as bridges between activates and basal transcription machinery

what are the roles of TAFs in TFIID? drosophila experiment

TBP- tata binding box protein associated factors: act as co-activators

• TFIID: TAFs and TBP

• adding TAF250 in drosophila→ no effecr

• TAF250 + TAF100→ increased transcription- bridging signalling for Bicoid

• TAF250+TAG60→increased transcription for Hunchback

• multiple TAFs→synergistic activation

TAFs act as co-activators

what are the roles of TAFs in TFIID? humans

• some TAFs associate with DNA- and act as cofactors

1. enable more efficient promotor recognition.

• when TFIIB binds- TAF1/2 bind to initator

• TAF6/9 downstream promtor elements

2. TAFs interact with other activators on the pre initiation complex

3. TAFS can have catalytic activity- TAF1 histone acetylation to open chromatin

4. chromatin recognition- can bind histone marker H3K4me→TAF3 which recruits TFIID

what is the mediator complex? what is its role in transcription?

• high conserved multi subunit

• essential for transcription activation- not just basal transcription

• doesn’t bind DNA directly, interacts with TFs

• bridge between enhancer-bound activators and RNA pol II

• stabilises pre initiation complex formation

• enhances transcription efficiency

• promotes dna LOOPING

• Co ordinates CTD phosphorylation in TFIIH regulation

26 core subunits

CDK8 kinase module- 4 subunits

recruited when transcription is activated- convert enhancer signals into PIC assembly and RNA pol II activation

how is the mediator complex organised? how does it interact t with transcription machinery?

1. head and middle

• interacts with RNA pol II and GTFs- for PIC recruitment and stabilisation

2. tail

• interacts with activator proteins at enhancers

3. CDK8 kinase module

• regulatory

• controls TF activity and transition to elongation

binds proteins- activators and POL II and GTFs!! NOT DNA

what is the mechanism of mediator complexes?

• mediator is recruited via activator and tail interactions by interaction with TFs

• DNA looping brings the enhancer to the promotor- together

• head/middle stabilise the interaction with the PIC and POL II

• CDK8 module dissociates during activation

• mediator promotes TFIIH H→CTD phosphorylation→ promtor escape

what is the upstream stimulatory activity complex? how does it regulate transcription? how does the positive USA work?

• multi protein regulatory complex- positive and negative cofactors- cancer enhance or repress. usually by blocking or promoting stable PIC assembly

pos PC4:

• binds DNA

• interacts with TFIIA and stabilises TBP binding to the TATA box

• bridges TFs- BRCA1, p53 etc

• promotes PIC assembly

• regulated by casein kinase II phosphorylation- phosphorylation reduces it

how do negative regulators in the upstream stimulatory activity complex work?

• NC1/HMG1- negative

• forms a stable complex with TBP and promtor DNA

• blocks TFIIB- can be overcome by TFIIA compeition

• NC2

• heterdimer lapha and beta

• binds TBP and stops TFIIA and TFIIB and the formation of DAB

• represses basal transcription

how do chromatin structures/remodeeling regulate TF access to DNA

1. nucleosomes- DNA around histone octamer- needs to be displaced to access TATA box

2. chromatin remodelling- SWI/SNF complex- recruited by transcription actuators ie SWI5 in yeast. has ATPase subunits BRG1 and BRM. remodel nucleosomes

• nucleosome is sliding along DNA and loop formation

• DNA exposure

• increases DNA accessibility for TFs and RNA pol II

3. histone proteins- chromatin modifying complexes HDACs and HATS

what does the SWI/SNF do?

• chromatin remodelling complex- yeast originally

• all eukaryotes

• 10+ protein complex- 4 core sununits

• changes nucleosome structure and/or position- makes it more accessible for transcription factors and RNA- helps unpack or reposition It so genes can be accessed

how does SWI/SNF work mechanistically?

• recruited by DNA bound activator proteins- SWI5

• activator binds dna→recruits SWI/SNF→chromatin rmeodelling

• uses ATP hydrolysis by BRG1/BRM- related to helicases, generate mechanical force to unwind

• ATP causes twisting/pushing- allows DNA to disengage from histones

outcomes of SWI/SNF activity

1. nucleosome sliding- moves along DNA and exposes previously hidden

2. transient DNA exposure- temporary loops so TF can bind temporarily

3. nculesome disassembly- H2A and then H2B- then H3/H4 is very strong

what are HATs how do they regulate transcription?

1. Nuetralise histone chagre- from pos to neutral. Less affinity for neg DNA

   • Doesn’t matter which- ACETYLATION means more open promotors and more transcription

   • Gcn5p- in the SAGA HAT complex- acetylate H3 and H2beta

   • P300- recruit to IFN Beta and acetylates 3 nucleosomes there

2. Creation of binding sites for specific proteins that recognise acetylated histones

   • Position of acetylation is crucial

   • Specific aa are acetylate don histones for binding sites.

     • Ie from signalling when tyrosine are phosphorylated can recruit sh2 DOMAINS which recognise phosphorylated domains

   • If you acetylate lysines- binding sequences for BROMO to bind to

   • TAF250: two bromodomains and reocgnise H4 acetylated lYS 5 AND LYS 12

3. Modulation of TF activity

• TFIIE, HMG proteins and p53 TF can be acetylated