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