Structural Studies of UBLCPI, a Novel CTD Phosphatase by NMR spectroscopy
description
Transcript of Structural Studies of UBLCPI, a Novel CTD Phosphatase by NMR spectroscopy
Structural Studies of UBLCPI, a Novel CTD Phosphatase by NMR
spectroscopy
연세대학교구조생화학 및 분자 생물리학 연구실
오효선
Introduction
Folded region : mRNA synthesis
Mobile CTD
RNA polymerase II- Mobile CTD 1) tail-like feature (52 heptapeptide repeats-YSPTSPS) 2) dynamic phosphrylation and dephosphorylation during transcription cycle 3) unphosphorylated CTD : trascription initiation hyperphosphorylated CTD : transcription elongation unphosphorylation need to recycle RNA polymerase II
Transcription and CTD phosphatase
UBLCP1 mutants
UBL domain
Phosphatase domain
1 80 133 294 318
Phosphatase domain
Phosphatase domain
UBL domain
100 318
133 318
801
Mutants : 1) Full structure (UBLCP1) 2) Phosphatase domain 3) Phosphatase domain + N-terminal (UBL PN)
UBL domain Phosphatase domain
1 80 133 294 318
M A L P I I V K W G G Q E Y S V T T L S E D D T V L D L K Q F L K T L T G V L P E R Q K L L G L K V K G K P A E N D V K L G A L K L K P N T K I M M M G T R E E S L E D V L G P P P D N D D V V N D F D I E D E V V E V E N R E E N L L K I S R R V K E Y K V E I L N P P R E G K K L L V L D V D Y T L F D H R S C A E T G V E L M R P Y L H E F L T S A Y E D Y D I V I W S A T N M K W I E A K M K E L G V S T N A N Y K I T F M L D S A A M I T V H T P R R G L I D V K P L G V I W G K F S E F Y S K K N T I M F D D I G R N F L M N P Q N G L K I R P F M K A H L N R D K D K E L L K L T Q Y L K E I A K L D D F L D L N H K Y W E R Y L S K K Q G Q
100
D I E D E V V E V E N R E E N L L K I S R R V K E Y K V E I L N P P R E G K K L L V L D V D Y T L F D H R S C A E T G V E L M R P Y L H E F L T S A Y E D Y D I V I W S A T N M K W I E A K M K E L G V S T N A N Y K I T F M L D S A A M I T V H T P R R G L I D V K P L G V I W G K F S E F Y S K K N T I M F D D I G R N F L M N P Q N G L K I R P F M K A H L N R D K D K E L L K L T Q Y L K E I A K L D D F L D L N H K Y W E R Y L S K K Q G Q
UBLCP1
Purification of UBLCP1
M 1 2 3 4 5 6 7 8 9
1. B/I 2. A/I 3. B/I 4. A/I5. Sup 6. ppt 7. F/T 8. W
9.elu
M 1 2 3 4 5 6
1. UBLCP12. TEV3. Second column
전4. F/T5. 25mM wash6. elution
53.79 kDa
36.8 kDa
2D 15N HSQC of UBLCP1
Phosphatase domain
20071113no007:10_UV 20071113no007:10_Conc 20071113no007:10_Fractions
0.0
10.0
20.0
30.0
40.0
50.0
60.0
mAu
0 20 40 60 80 100 ml
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
< sperdex75 column >
Molecular weight :25.9kDa
Purification of phosphatase domain
M 1 2 3 4 5 6 7
Trx-(His)6 tag fusion
phosphatase (41.4kDa)
Trx-(His)6-tag
Phosphatase(25.9kDa)
← 2D 1H-15N HSQC spectrum (pH 6.0)
2D 1H-15N HSQC spectrum → (pH 8.5)
2D 1H-15N HSQC spectrum (pH 6.5)
Acknowledgement
Yonsei univ.
Prof. Weontae LeeDr. Sunggeon KoSooyoung KwakYongmin Lee
Prof. Jong-Bok Yoon