20.109:TAP-tags

New Protein Engineering Module for F08

Point of departure is to ask if tags are actually neutral wrt protein function

Part 1: TAP tag
- non-essential SAGA subunits (essential likely in many complexes so purif profile complex + tag has many consequences)
- unknown ORFs changed during microarray of SAGA del or mtRNT1
  - 1-1 Design primers
  - 1-2 PCR
  - 1-3 Transform
  - 1-4 Confirm integration by colony PCR
- Alternatively, could just give students tagged version.
Part 2: Western
- need + TAP control
- use Sigma Ab to protA (anti-ProtA raised in rabbit or if need to detect tag when protA has been cleaved can use OpenBiosystems Ab)
- 1.5 ml ON culture, vortex 3X30' with glass beads and add 100 ul LB from Methods (2001) 24:218 barton link
Part 3: Phenotypes
- compare parental, K/O, TAP-tagged
- examine GAL, Spt (his4-917d), TS, CS
Part 4: Array
- compare parental to TAP-tagged
Could also include
- Mass Spec analysis description/tour/lab?
- Tour of protein purif facility

FB2305
- MC1066+pBS1539 (AmpR+URA3-Kl, TAP tag)
FB2306
- MC1066+pBS1479(AmpR+TRP1-Kl, TAP tag)
- ref for seq of TRP1 from Kluyveromyces lactis here
- NCBI link for seq of TRP1 from Kluyveromyces lactis here

FY631 = Parental for TAP tagging
- MATa, his4-917d, lys2-173R2, leu2d1, ura3-52, trp1d63
FY2714
- MATa, HA-SPT3-TAP::TRP1, his4-917d, lys2-173R2, ura3D0 or -52, leu2D1 or D0, trp1D63
FY2031
- MATa, HA-SPT7-TAP::TRP1, his4-917d, lys2-173R2, ura3D0, leu2D1, trp1D63
FY2305
- MATa, SPT10-TAP::TRP1 trp1D63 his3D200 leu2D1 ura3-52 lys2-128d
- not his4-912d
FY2306
- MATa, SPT21-TAP::TRP1 trp1D63 his3D200 leu2D1 ura3-52 lys2-128d
- not his4-912d

needed to	forward primer (5' to 3')	reverse primer (5' to 3'
add TAP TRP to C-term of unknown 8	GGT ATT TGT CGA TTA CAA AAC ACA TCC TGT AGG CGC AAA TTC CAT GGA AAA GAG AAG 57 mer Tm = 66.7	GTT AGG ATT CAG TAC TAA CAC ATT CTC TAT GAC ACA ACC TTA CGA CTC ACT ATA GGG 57 mer Tm = 65.0
check TAP	TCC ATG GAA AAG AGA AGA TGG AAA AAG AAT TTC ATA GCC GTC TC 44 mer 63.6 use with reverse primer from tagging PCR expect length of TAP seq + TRP K. l seq =	TCA GGT TGA CTT CCC CGC GGA ATT CGC GTC TAC 33 mer 67.9 use with fwd primer from tagging PCR
add TAP TRP to C-term of RNT1	CTC ACA AAA GAA TAA GAA AAG AAA ATT CTC AGA TAC AAG CTC CAT GGA AAA GAG AAG 57 mer Tm = 63.8

Subunit	size,chromosome,null p-type

Ada subunits

Ada1 (aka HFI1, SUP110, SRM12, GAN1)	1.467 kb=489 aa, Chr. XVI, viable
Ada2 (aka SWI8)	1.305 kb=434aa, Chr. IV, viable
Ada3(aka NGG1, SWI7)	2.109 kb=702aa, Chr. IV, viable
Gcn5 (aka ADA4, SWI9)	1.32 kb=439aa, Chr. VII, viable
Ada5 (aka SPT20)	1.815 kb=604aa, Chr. XV, viable

Spt subunits

Spt3	1.014 kb=337aa, Chr. IV, viable
Spt7(aka GIT2)	3.999 kb=1332aa, Chr. II, viable
Spt8	1.809 kb=602aa, Chr. XII, viable
Spt20 (aka Ada5)	1.815 kb=604aa, Chr. XV, viable

TAF subunits

TAF5 (aka TAF90)	2.397 kb=798aa, Chr. II, inviable
TAF6 (aka TAF60)	1.551 kb=516aa, Chr. VII, inviable
TAF9 (aka TAF17)	0.474 kb=157aa, Chr. XIII, inviable
TAF10 (aka TAF23, TAF25)	0.621 kb=206aa, Chr. IV, inviable
TAF12(aka TAF61, TAF68)	1.620 kb=539aa, Chr. IV, inviable

Tra subunit

Tra1	11.235 kb=3744aa, Chr. VIII, inviable

Other subunits

Sgf73	1.974 kb=657aa, Chr. VII , viable
Sgf29	0.779 kb=259aa, Chr. III, viable
Sgf11	0.3 kb=99aa, Chr.XVI, viable
Ubp8	1.416 kb=471aa, Chr. XIII, viable
Sus1	gene with intron, Chr. II, viable

Unknown	ORF	SGF73 green signal	sgf73 red signal	log2 (green/red)
1	YHR033W	38938 and 69586	285 and 570	7.1 and 6.9
2	YOR302W	3374 and 6054	49 and 167	6.1 and 5.2
3	YJR097W	524 and 1052	13 and 28	5.3 and 5.2
4	YBL028C	1146 and 2706	32 and 323	5.2 and 3.1
5	YDR034W-B	17290	447	5.3
6	YGR067C	12025	320	5.2
7	YKL037W	8340	282	4.9
8	YER067W	6296 and 12450	82556 and 81036	-3.7 and -2.7