20.109:TAP-tags

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Contents

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
  • 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]
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 [2]
from [3]
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
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