BIOL 2010 - Control of transcription in eukaryotes

types of polymerase

POL I

• transcribes ribosomal RNA

POL II

• transcribes genes that encode proteins and most snRNA

POL III

• transcribes tRNA, some RNA and other small RNA

transcription factors:

IMPORTANCE

• determine whether transcription occurs

• determine cell specificity

• confer responses to specific stimuli

ISOLATION OF TFS

• to find TFs: bioinformatic analysis of the whole genome and look for conserved regions

STRUCTURE OF TFS

• TFs have a modular structure

  • one region binds to DNA

  • another region binds to the components

• all the AAs for DNA binding is found in one region

  • oct 2 has 60AAs region which binds to DNA —> this 60AA region has the same affinity as the full length of Oct 2

• the other region binds to other things either bound to activation domains or inhibitor domains (sometimes this binding is direct, sometimes through intermediate cofactors)

ISOLATION OF TFS

• DNA sequence encoding TF is synthesised and bound to beads

• proteins are extracted from lysed cells and combined with the beads

• the TF protein will bind to binding sequence so will stick to the beads

• protein can then be extracted from the bead

classes of DNA binding domains

most Tfs have 1 of these 3 binding domains

these are different strategies of proteins for inserting themselves into DNA so that they fit tightly

inserted into the major groove due to charge, size, shape etc

1. zinc finger

2. helix turn helix

3. basic binding domains

1. Zinc finger

• protein that contains an alpha helic and Zn2+ which keeps all of the AAs in shape

• usually attached in a multimeric way

EXAMPLE: Sp1TF

• contains a loop of 23 AAs

• link between multiple zinc fingers is 7-8 AAs

• alpha helix contains major groove

• specifically driven by multiple zinc fingers

• Zn2+ doesnt directly interact with the DNA but is essential for the folding of the finger

• zinc fingers bind both the major and the minor grooves

2. Helix turn helix

• alpha helix, turn followed by another helix

• one is a stabilising helix and the other is a recognition helix

• multiple helixes and turns —> most commonly 3

• E.g: homeobox TFs

3. basic binding domain

• TFs w/ basic binding domain cant bind alone so they must dimerise

• form 2 prongs that can straddle the DNA helix

• E.g leucine zipper or helix loop helix

regulation of TFs

• may be regulated by location if responsiveness is important

• E.g: steroid hormone receptors (they have a zinc finger binding domain)

• E.g: vit D receptors

VIT D RECEPTOR

• Vit D receptor in the cytoplasm

• binding of vit D to receptor causes dimerization and translocation into the nucleus

• in the nucleus it binds to a consensus sequence which causes transcription of VD responsive genes

how do transcription factors activate transcription?

• binding region alone doesnt cause transcription

• activation/repressor domain is responsible for transcription

• to determine which region is the activator/repressor you can take a well known TFs binding domain and add on parts of an unknown TF which you suspect may be an activation domain

• whichever one causes transcription will be the activation domain

regulation of elongation

• regulated by promoters upstream of the core promoter

• cell fate determined by gene expression —> regulated by TFs

• this control is controlled by tissue specific TFs:

UPSTREAM SEQUENCE ELEMENTS:

• can enhance the binding of PIC to increase transcription of a gene

• regulator proteins binding affects affinity of RNA pol to promoter

• E.g: GC box and CAT box

• upstream sequence elements present in housekeeping genes

• must be in the same orientation as the gene to be effective

• E.g: myo D is a gene found in all cells but is only expressed in muscle cells

enhancer/activator regions

• usually a long way upstream/downstream of the gene

• not part of the promoter but works in a similar way

• bound by activator proteins

• DNA structure means that the enhancers are close to the promoter due to the way it folds

• can be any orientation (5’—>3’ or 3’—>5’)

• can also be found in the introns so mutations in the introns will still affect the phenotype

different types of activation/enhancer domains

• only have generalised properties, a lot less well defined than the categories of binding domains

• activation domains interact with a whole host of proteins depending on the function of the Tfs rather than the binding domains which fit a specific sequence of DNA

  1. Acidic AAs

  2. Glutamine rich

  3. proline rich

how do activation/enhancer domains work?

• the pre initiation complex is assembled

• the general TFs occupy so much space that the activator protein and regulatory proteins are able to bind to structures of DNA on the opposite sides of the promoter

• may cause a conf change in the PIC which is more tightly bound which enables transcription

• or through the recruitment of coactivators:

  • coactivators work by interacting with the PIC

  • sometimes they work by the interaction w the DNA by loosening or opening the chromatin structure to allow the PIC to bind better to the DNA

how do activators alter chromatin structure

• co activators can modify histones

• histones have 2 domains

  • globular domain

  • amino tail domain rich in lysine

• if the coactivator has histone acetyltransferase activity then they can neutralise the +ve charge of the lysine which causes the histone to open up as the DNA no longer attracted to the +ve charge of the histone

• transcription can occur more readily

• E.g glucocorticoid receptor can recruit co activator p300/CBP which has HAT activity

inhibitory domains

• not done in whole blocks of genes, only works on an individual gene basis

• inhibitory domains bind to DNA in the same way as the activating domains and they block TFs with activator domains from binding

• alternatively, they might bind directly to the PIC and reduce the affinity of the PIC for the core promoter

• both these methods work by physically getting in the way but they can also work through co repressors:

CO REPRESSORS:

• indirect interaction between PIC and the repressor

• opposite of the activation —> close the DNA structure by HDAC (histone deacetylase) removing the acetyl group of histone units which restores the positive charge of the histone, preventing transcription

EXAMPLES:

1. SMRT

   • forms a large complex w/ RAR or transcriptional repressors

   • has HDAC capability

2. tamoxifen

   • oestrogen is important in breast cancer development

   • tamoxifen is a drug that enhances the corepressors to the oestrogen receptor —> prevents proliferation of oestrogen dependent cancers

   • however cancers can become tamoxifen resistant