Eukaryotic Transcription II

Proteins that control RNAPII initiation at eukaryotic promoter/initiation sites

Basal txn factors

How does RNAP initiate in eukaryotes?

-          if add pure RNAP to DNA containing a promoter, don’t get specific initiation

o       if add E coli RNAP to DNA containing a promoter, do get

-          how to make it initiate specifically?

-          Make nuclear extract, add to RNAP + DNA, now get specific initiation (Dignam and Roeder!)

-          Fractionate extracts

-          Must add several different fractions to get specific initiation

-          Named TFII A,B,D etc

-          Now known to be protein complexes (general transcription factors)

-          TFIID has a subunit that binds the TATA box (TBP)

o       Also has other subunits, called TAFs

-          others don’t bind DNA, but seem to nucleate on TFIID

o       one picture IID, IIB, IIF+RNAPII (now we have finally positioned RNAPII at the promoter), IIE, finally IIH

o       TFIIH has helicase activity – unwinds duplex in local region

o       Somehow, buildup of all these general transcription factors makes RNAPII initiate txn

Activator proteins

-          bind to proximal promoter elements and enhancers

-          have a modular organization with at least two domains

o       DNA binding domain

§         Homeodomains (helix-turn-helix)

·         named because first found in homeotic genes, which transform one segment of a fly to another segment

·         these genes encode transcription factors

·         their binding domain is always a helix-turn-helix

§         Zinc finger domains

·         C₂H₂ have two cysteines and two histidines coordinating Zn²⁺ ion

o       Proteins often will have several of these, each contacting a few bp

·         C₄ have four cysteines

o       Steroid hormone receptors and a few others

o       Usually have two fingers

·         Actual structure has some b–sheet and a helix which binds DNA in major groove

§         basic helix-loop-helix (bHLH)

·         helix that binds DNA, then loop, then a long helix used for dimerization

o       transcriptional activation domain

§         much less in common

§         one common finding, rich in amino acids with acidic side-chains (Asp and Glu)

§         negatively charged at pH7

§         famous experiment, 1% of random DNA (from Ecoli) fused to GAL4 DNA binding domain function as activators

·         only thing in common is all acidic

·         “acid blob”

o       Hormone binding domain

§         Steroid hormone receptors are regulated txn factors

§         Have the above two domains, plus a hormone binding domain that regulates when they work

·         Can regulate when they enter the nucleus

·         Can regulate activity when already in nucleus

-          Dimerization allows increased specificity of control

o       can form homo- or heterodimers with other bZIP or bHLH partners

o       only dimeric form can bind DNA, activate transcription

o       can even partner with a monomer that doesn’t bind DNA, preventing DNA binding by heterodimer

Repressor proteins

-          repress txn

o       different mechanisms

§         bind DNA sequence and prevent activator from binding

§         bind to activator protein and

·         prevent from binding DNA

·         prevent from activating txn

GAL txn system

-          need three genes for three proteins for cell to metabolize galactose

o       GAL1

o       GAL7

o       GAL10

-          Need all on or all off

-          Do regulated txn by putting the same enhancer upstream of all of these genes

o       Activator protein that binds is called GAL4p

-          How is txn regulated?

o       In glucose, GAL4p is not around, so no binding

o       In raffinose, GAL4p bound, but repressor GAL80 is covering up activation domain, so no txn

o       In galactose, GAL1 or GAL3p bind galactose, change shape, and bind to GAL80, changing its binding to GAL4 so that activation domain of GAL4 is exposed

Myc-Max system

-          myc looks like a bHLH activator protein, but cannot bind DNA on its own

-          max has DNA binding and dimerization domains, but no activation domain

-          max-max make homodimer which binds enhancers, represses txn

-          myc-max make heterodimers which bind enhancer and activates txn

o       max has higher affinity for myc than myc, so when both are present get myc-max heterodimers

-          cells not proliferating only make max

-          when cells want to proliferate, make myc, turn on a bunch of genes that stimulate the cell to proliferate

-          myc is an oncogene – it is mutated in cancer cells so that it always on, thus always stimulating cell to proliferate