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Structural Studies of UBLCPI, a Novel CTD Phosphatase by NMR spectroscopy

Structural Studies of UBLCPI, a Novel CTD Phosphatase by NMR spectroscopy. 연세대학교 구조생화학 및 분자 생물리학 연구실 오효선. Introduction. Transcription and CTD phosphatase. Folded region : mRNA synthesis. RNA polymerase II - Mobile CTD 1) tail-like feature (52 heptapeptide repeats-YSPTSPS)

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Structural Studies of UBLCPI, a Novel CTD Phosphatase by NMR spectroscopy

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  1. Structural Studies of UBLCPI, a Novel CTD Phosphatase by NMR spectroscopy 연세대학교 구조생화학 및 분자 생물리학 연구실 오효선

  2. Introduction

  3. Transcription and CTD phosphatase Folded region : mRNA synthesis 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 Mobile CTD

  4. 1 133 294 318 80 UBL domain Phosphatase domain UBL domain 1 80 Phosphatase domain 133 318 100 318 Phosphatase domain UBLCP1 mutants Mutants : 1) Full structure (UBLCP1) 2) Phosphatase domain 3) Phosphatase domain + N-terminal (UBL PN)

  5. 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 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 1 133 294 318 80 UBL domain Phosphatase domain 100

  6. UBLCP1

  7. M 1 2 3 4 5 6 M 1 2 3 4 5 6 7 8 9 Purification of UBLCP1 • UBLCP1 • TEV • Second column전 • F/T • 25mM wash • elution 36.8 kDa • B/I 2. A/I 3. B/I 4. A/I • 5. Sup 6. ppt 7. F/T 8. W 9.elu 53.79 kDa

  8. 2D 15N HSQC of UBLCP1

  9. Phosphatase domain

  10. M 1 2 3 4 5 6 7 Trx-(His)6 tag fusion phosphatase (41.4kDa) Phosphatase(25.9kDa) Trx-(His)6-tag < sperdex75 column > Molecular weight :25.9kDa Purification of phosphatase domain

  11. ← 2D 1H-15N HSQC spectrum (pH 6.0) 2D 1H-15N HSQC spectrum → (pH 8.5)

  12. 2D 1H-15N HSQC spectrum (pH 6.5)

  13. Acknowledgement Yonsei univ. Prof. Weontae Lee Dr. Sunggeon Ko Sooyoung Kwak Yongmin Lee Prof. Jong-Bok Yoon

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