20.109:TAP-tags

New Protein Engineering Module for F08

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

Rough sketch of labs

• 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

Experimental Details

Bacteria

• FB2305 = NB 311
  • MC1066+pBS1539 (AmpR+URA3-Kl, TAP tag)
• FB2306 = NB312
  • 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

from Methods 2001 article[1]

• NB318
  • XL1-bl+pBS1479 (Lisa's prep of plasmid which does give PCR product for TAP-TRP, freshly transformed)

Yeast

• FY631 = Parental for TAP tagging = NY411
  • MATa, his4-917d, lys2-173R2, leu2d1, ura3-52, trp1d63
• FY2714 = NY412
  • MATa, HA-SPT3-TAP::TRP1, his4-917d, lys2-173R2, ura3D0 or -52, leu2D1 or D0, trp1D63
• FY2031 = NY413
  • MATa, HA-SPT7-TAP::TRP1, his4-917d, lys2-173R2, ura3D0, leu2D1, trp1D63
• FY2305 = NY414
  • MATa, SPT10-TAP::TRP1 trp1D63 his3D200 leu2D1 ura3-52 lys2-128d
  • not his4-912d
• FY2306 = NY415
  • MATa, SPT21-TAP::TRP1 trp1D63 his3D200 leu2D1 ura3-52 lys2-128d
  • not his4-912d

Primers for C-terminal fusions

General format for

• Upstream primer: 40nt then TCC ATG GAA AAG AGA AG
• Downstream primer: 40nt then TAC GAC TCA CTA TAG GG

Relevant sequence files

1. sequence of TAP tag only here
2. sequence of pBS1479 here

from [2]

from [3]

Needed primers

needed to	forward primer (5' to 3')	reverse primer (5' to 3'
add TAP TRP to C-term of unknown 8	NO222 GGT ATT TGT CGA TTA CAA AAC ACA TCC TGT AGG CGC AAA TTC CAT GGA AAA GAG AAG 57 mer Tm = 66.7 landing Tm (17bases only) = 45.5	NO223 GTT AGG ATT CAG TAC TAA CAC ATT CTC TAT GAC ACA ACC TTA CGA CTC ACT ATA GGG 57 mer Tm = 65.0 landing Tm (17bases only)= 46.1
check TAP	NO224 TCC ATG GAA AAG AGA AGA TGG AAA AAG AAT TTC ATA GCC GTC TC 44 mer Tm = 63.6 use with reverse primer from tagging PCR	NO225 TCA GGT TGA CTT CCC CGC GGA ATT CGC GTC TAC 33 mer Tm = 67.9 use with fwd primer from tagging PCR expect length of TAP seq + TRP K. l seq = 554 bps (from 2147 to 2701 in pBS1479)
add TAP TRP to C-term of RNT1	NO226 CTC ACA AAA GAA TAA GAA AAG AAA ATT CTC AGA TAC AAG CTC CAT GGA AAA GAG AAG 57 mer Tm = 63.8	NO227 ATC AAT GCA AGT TCC ATC ATG GTT GTG TAA AAG GAA CGT TTA CGA CTC ACT ATA GGG 57 mer Tm = 66.9
longer landing sequences	TCC ATG GAA AAG AGA AGA TG = 20 mer fwd landing Tm = 49.6 ºC	TAC GAC TCA CTA TAG GGC GA = 20 mer rev landing Tm = 54.3 ºC
Unknown 8 + longer landing sequences	NO228 GT ATT TGT CGA TTA CAA AAC ACA TCC TGT AGG CGC AAA TTC CAT GGA AAA GAG AAG ATG 59 mer Tm = 66.5 ºC	NO229 TT AGG ATT CAG TAC TAA CAC ATT CTC TAT GAC ACA ACC TTA CGA CTC ACT ATA GGG CGA 59 mer Tm = 66.6 ºC
RNT1 + longer landing sequences	NO230 TCACAA AAGAATAAGA AAAGAAAATT CTCAGATACA AGC TCC ATG GAA AAG AGA AGA TG 59 mer Tm = 64.4 ºC	NO231 TCA ATG CAA GTT CCA TCA TGG TTG TGT AAA AGG AAC GTT TAC GAC TCA CTA TAG GGC GA 59 mer Tm = 68.4 ºC
check TAP	NO258 for SPT3-TAP control TTT AAA GGT GGT AGA CTC AGT TCT AAA CCA ATT ATC ATG tcc atg gaa aag aga aga tg 59 mer use with NO225 = reverse primer for checking TAP tag expect length of TAP seq + TRP K. l seq = 554 bps (from 2147 to 2701 in pBS1479)	NO259for SPT7-TAP control AAT AGT TCA TTT AGC TTG AGC CTT CCT CGC CTT AAT CAA tcc atg gaa aag aga aga tg 59 mer use with NO225 = reverse primer for checking TAP tag expect length of TAP seq + TRP K. l seq = 554 bps (from 2147 to 2701 in pBS1479)
Check TAP with NO224	SPT3_check_rev NO268 ACC AGA AGG AAA CCC ATG CAC CTC CAT GAT GAA ATT ATA CAA AAA T	SPT7_check_rev NO269 AGC AAA TGT ATT TAA TTA AAG ATA AAA TAT TCA ACT ATT TAG CGC GCT CA

Genes to tag

Option 1: S. cerevisiae SAGA subunits

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

Option 2: Unknown ORFs from SGF73/sgf73 array

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