User:Nkuldell/SAGA swap: Difference between revisions
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| TAF9 [[http://db.yeastgenome.org/cgi-bin/locus.pl?locus=TAF9]] | | TAF9 (aka TAF17) [[http://db.yeastgenome.org/cgi-bin/locus.pl?locus=TAF9]] | ||
| 0.474 kb, Chr. XIII, <font color = red>inviable</font color> | | 0.474 kb, Chr. XIII, <font color = red>inviable</font color> | ||
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===S. cerevisiae vs human SAGA subunits=== | ===S. cerevisiae vs human SAGA subunits=== | ||
====1. structures==== | |||
*EM of human TFTC in [[Image:Macintosh HD-Users-nkuldell-Desktop-TFTCvsSAGA Sci99.pdf]] | *EM of human TFTC in [[Image:Macintosh HD-Users-nkuldell-Desktop-TFTCvsSAGA Sci99.pdf]] | ||
*EM of ySAGA in [[Image:Macintosh HD-Users-nkuldell-Desktop-SAGA swap-SAGAstruct MolCell05.pdf]] | *EM of ySAGA in [[Image:Macintosh HD-Users-nkuldell-Desktop-SAGA swap-SAGAstruct MolCell05.pdf]] | ||
====common subunits==== | ====2. common subunits==== | ||
{| border="1" | {| border="1" | ||
! subunit | ! subunit | ||
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| TAF6 (aka TAF60) | |||
| [[http://db.yeastgenome.org/cgi-bin/locus.pl?locus=TAF6]] | |||
| 1.551 kb, Chr. VII, <font color = red>inviable</font color> | |||
| hTAF80 | |||
| | |||
| | |||
|} | |} | ||
human TFTC-specific subunits | human TFTC-specific subunits (from Wu in Mol Cell 04) | ||
*PAF65-beta | *PAF65-beta | ||
*Taf2 | *Taf2 | ||
*Taf4 | *Taf4 |
Revision as of 13:59, 3 June 2006
Idea
Justification
The genetic and epigenetic requirements for gene expression will need to be considered if logic circuits and genetic programs are to be reliably written for eukaryotic cells. Since chromatin dynamics are an integral part of eukaryotic gene regulation, a better understanding of chromatin modifying complexes will be necessary if DNA sequences are to preform, i.e. express, in a predictable way. Ideally a common chromatin remodelling complex could be described that would perform seemlessly in any (or less ambitiously, several) hosts/operating system. Is there a generic complex that could plug into multiple eukaryotic hosts and properly regulate histone acetylation/deacetylation as well as nucleosome positioning over transcribed regions? As a first step in designing such a common chromatin remodeller, the SAGA subunits of S. cerevisiae can be replaced with the S. pombe homologs. Shoot, if Apple can find a way to run a MS operating system, smart yeast should make this SAGA swap possible.
Proposal Details
Standard yeast techniques can be used to replace the S. cerevisiae subunits with the homologs from S. pombe and effects on gene expression can be assessed. Specifically it should be possible to integrate a URA3 marker at a gene encoding a SAGA-subunit and then replace that marker wtih the S. pombe gene by transformation and seletion on FOA. The pombe genes would be amplified from a cDNA library, the source of which is still to be determined. Expression of each replaced subunit can be followed by Western if Abs are available or with epitope tags (less desirable), SAGA integrity can be functionally and biochemically assessed, cell-wide effects on gene expression can be followed by array, mutant phenotypes (Spt, drug sens etc...) Single gene replacements of multisubunit are often unsuccessful but activity can be restored with multiple replacements of interacting subunits (check Stan Fields STE12 and MCM1?)
Four distinct classes of genes make up the multisubunit SAGA complex in S. cerevisiae
1. The Ada proteins
- Ada1, Ada2, Ada3, Gcn5, Ada5
2. The Spt proteins
- Spt3, Spt7, Spt8, Spt20
3. The TAF proteins
- TAF5, TAF6, TAF9, TAF10, TAF12
4. The Tra1 protein
- essential gene
- target of gene specific activators
Can generic versions of these subcomplexes be described and then used to intelligently specify the chromatin packaging needed to execute a genetic program? Success would provide an existance proof for a re-usable genetic module capable of appropriately remodelling chromatin in N>1 host.
Fact tables
S. cerevisiae SAGA chemistry
summarized from TiBS 05 review File:Macintosh HD-Users-nkuldell-Desktop-SAGA swap-SAGAunveiled TiBS05.pdf
- GCN5 has HAT activity
- Ada2, Ada3 regulate GCN5
- Ada5 is = Spt20
- SAGA structural integrity depends on Ada1, Spt7, Spt20, and TAF12
- interaction with TBP requires Spt8 and Ada3
- gene specific transcriptional activators Gcn4, VP16 and Gal4 target Tra1
- histone fold pairs: TAF6 and TAF9, TAF10 and Spt7, TAF12 and Ada1
- SAGA variants such as SALSA, SLIK without Spt8 and truncation of Spt7/ of questionable functional significance
S. cerevisiae vs S. pombe SAGA subunits
Ada subunits | cerevisiae | gene size,chromosome,null p-type | pombe | gene size,chromosome,null p-type | BLAST comparison info |
---|---|---|---|---|---|
Ada1 [[1]] | 1.467 kb, Chr. XVI, viable | ||||
Ada2 [[2]] | 1.305 kb, Chr. IV, viable | ||||
Ada3 [[3]] | 2.109 kb, Chr. IV, viable | ||||
Gcn5 [[4]] | 1.32 kb, Chr. VII, viable | ||||
Ada5 [[5]] | 1.815 kb, Chr. XV, viable |
Spt subunits | cerevisiae | gene size, chromosome, null p-type | pombe | gene size, chromosome, null p-type | BLAST comparison info |
---|---|---|---|---|---|
Spt3 [[6]] | 1.014 kb, Chr. IV, viable | ||||
Spt7 [[7]] | 3.999 kb, Chr. II, viable | ||||
Spt8 [[8]] | 1.809 kb, Chr. XII, viable | ||||
Spt20 [[9]] | same as Ada5 |
TAF subunits | cerevisiae | gene size, chromosome, null p-type | pombe | gene size, chromosome, null p-type | BLAST comparison info |
---|---|---|---|---|---|
TAF5 (aka TAF90) [[10]] | 2.397 kb, Chr. II, inviable | spTAF72 | |||
TAF6 (aka TAF60) [[11]] | 1.551 kb, Chr. VII, inviable | spTAF50 (akaSPCC16C.18c) | inviable | 39% identical File:Macintosh HD-Users-nkuldell-Desktop-ScSpTAFs NAR02.pdf | |
TAF9 (aka TAF17) [[12]] | 0.474 kb, Chr. XIII, inviable | ||||
TAF10 [[13]] | 0.621 kb, Chr. IV, inviable | ||||
TAF12 [[14]] | 1.620 kb, Chr. IV, inviable |
Tra1 subunit | cerevisiae | gene size, chromosome, null p-type | pombe | gene size, chromosome, null p-type | BLAST comparison info |
---|---|---|---|---|---|
Tra1 [[15]] | 11.235 kb, Chr. VIII, inviable | hypotheical protein SPBP16F5.03c [[16]] | Chr. II | GeneDB [[17]] |
S. cerevisiae vs human SAGA subunits
1. structures
- EM of human TFTC in File:Macintosh HD-Users-nkuldell-Desktop-TFTCvsSAGA Sci99.pdf
- EM of ySAGA in File:Macintosh HD-Users-nkuldell-Desktop-SAGA swap-SAGAstruct MolCell05.pdf
2. common subunits
subunit | cerevisiae | gene size, chromosome, null p-type | human | gene size, chromosome, null p-type | BLAST comparison info |
---|---|---|---|---|---|
Spt7 | [[18]] | 3.999 kb, Chr. II, viable | |||
TAF6 (aka TAF60) | [[19]] | 1.551 kb, Chr. VII, inviable | hTAF80 |
human TFTC-specific subunits (from Wu in Mol Cell 04)
- PAF65-beta
- Taf2
- Taf4