1. Group meeting Mahavir Singh 01/19/2010 Tetrahymena p65 And Shq1, a H/ACA related protein 1

2. Stone MD et al. Nature. 2007; 22;446(7134):458-61. Teixeira MT, and Gilson E. NSMB 2007; 14(4):261-2. p65 p65TERT Tetrahymena telomerase holoenzyme assembly Part I: Tetrahymena telomerase assembly protein p65 Richards RJ et al. RNA 2006; 12: 1475-1485 p45 p75 p19 2 Min B and Collins K, Mol Cell, 2009 N-termLaRRMC-term 1542 1 1100 T2 CP2 CPT1 2 A B`B` CD E t p65 t TERT TEN (1-195)RBD (195-516) p50 Catalytic core

3. Outline NMR: - Structure determination of nCter02 (369-519) and Cter01dL (369ΔL542 ). - nCter01 (369-542)/pstem4 and Cter01dL (369ΔL542)/pstem4 complex. Crystallization: - Crystallization attempts on p65/TER RNA complexes (with and without U1A). Full length p65: N-termLaC-ter 1 542 pC01 RRM Cter constructs: nCter01 (369-542) nCter02 (369-519) nCter06 (358-542) nCter10 (310-542) nCter02_short (443-542) Cter02dL (369ΔL519 ) Cter01dL (369ΔL542 ) RRM and Cter (RC) constructs: RC01 (213-542) RC02 (247-542) RC03 (236-542) Full length (FL) constructs: FL (1-542) FL01 (19-542) FL02 (50-542) FL03 (87-542) NMR Crystallization

4. Part 1: NMR studies of p65-Cter -Cter02 (369-519) and Cter02dL (369ΔL519). -Interaction of nCter01 (369-542) andCter01dL (369ΔL542) with pstem4 RNA.

5. nCter02 (369-519) assignment: In collabration with BkKoo Backbone assignment: >90% Side chain: ~80% Previous results 369 519

6. 1H-15N hetnoe/backbone dynamics (nCter02): what parts of protein are flexible 369 519 369 519 421 442 Previous results

7. NMR of nCter02dL (369ΔL542 ) construct 369 519 369 519 421 442 Previous results

8. NMR structure determination of Cter02dL (369ΔL519 ) Experiments Assignment progress 2D 1H-15N HSQCAssigned, in progress 2D 1H-13C HSQC-- 3D HNCBCA/CBCACONHAssigned 3D HNCA/HNCOCAAssigned 3D HNCO/HNCACOAssigned 3D CCCONHAssignment in progress 3D HCCCONHDid not work well 3D 15N TOCSY-HSQCDid not work well 3D HBHACONHAssigned 3D HNHARecorded 3D HCCH-TOCSYAssigned 3D 15N NOSEY-HSQC (mixing time 120 and 100 msec) Assignment under progress 3D 13C NOSEY-HSQC (mixing time 120 and 100 msec) Assignment under progress Total assignment is ~82% including ~99% backbone and rest side chain assignment!

9. Cter02dL Cter02dL (369ΔL519 ) assignment: Missing backbone assignment: L480, N482 Improvement in spectra Cter02dL vs. sHCter02 An example:

10. N C Lowest energy structure Preliminary structure of Cter02dL (369ΔL519 ) α1 Β sheet 1 N C α2 α3 Β sheet 2 S1S2S3 S4 S5 RNA binding part C loop Statics Rmsd (370-415, 445-516): 4.5 Total noe: 1071 Rmsd to the mean, helix 1: 1.40 Rmsd to the mean, helix 2: 1.57 Rmsd to the mean, helix 3: 1.64 Rmsd to the mean, sheet 1: 1.5 Rmsd to the mean, sheet 2: 2.17

11. Cter02dL (369ΔL519 ) structure update a =  -helix b =  -strand Problems: Large unstructured/flexible region in the middle of the protein (residues 417-451). Overlap for the longs side-chain amino acids: Ile:17 Leu:12 Asn:15 Gln:13 Lys:10 Glu:10 No sequence homology with other protein. Incomplete aromatic assignment. N C loop

12. Is Cter02dL (369ΔL519 ) an non-canonical RRM? View 1 View 2 p65-Cter does not show any significant sequence homology with any protein in BLAST search! Structural homology with RRMs? View 3 View 4 Clery, Blatter and Allain, Cur. Op. Str. Biol, 2008 RRM1

13. nCter01 (369-542) vs. pstem4 titration Protein: RNA :: (1:0) Protein: RNA :(1:1.25) Buffer: 20 mM Pi, 50 mM NaCl, pH 7, 2 mM DTT @ 800 MHz @ 35 deg (U14) (U28) (U30) (U22) (G23) (G27) (G3) (G20) (G19) U16 Previous result 369 542

14. sHnCter01 (360-542) vs. Cter01dL (369ΔL542 ) 369 542 369 542 421442 Buffer: 20 mM Pi, 50 mM NaCl, pH 7, 1 mM DTT @ 800 MHz @ 25 deg

15. Cter01dL (369ΔL542 ) vs. pstem4 titrations 369 542 421442 Protein:RNA 1:0 Protein:RNA 1:0.5 Protein:RNA 1:0.75 Protein:RNA 1:1 Protein:RNA 1:1.25 Protein:RNA 1:2 Buffer: 20 mM Pi, 50 mM NaCl, pH 7, 1 mM DTT @ 800 MHz @ 25 deg

16. Cter01dL (369ΔL542 ) vs. pstem4 titration (U14) (U28) (U30) (U22) (G23) (G27) (G3) (G20) (G19) U16 Buffer: 20 mM Pi, 50 mM NaCl, pH 7, 1 mM DTT @ 800 MHz @ 25 deg 369 542 421442 Protein:RNA 1:1.25

17. Cter01dL (369ΔL542 ) vs. Cter02dL (369ΔL519 ) 369 519 421 442 369 542 421442

18. Part 2: crystallization of p65 FL w/wo RNA -Cloning and purification of TER_U1A constructs -Purification of proteins: p65 FL and U1A -Setting crystals of the RNA-U1A, RNA-p65, and RNA- U1A-p65 ternary complex

19. p65 constructs Salt Crystals!!!!! Previous results

20. TER_U1A Constructs U1A Tetrahymena telomerase pre-holoenzyme complex p65 GCGC GCGC Mfold prediction

21. TER_U1A Constructs TER_U1A00 TER_U1A01 TER_U1A02 U1A ✔ ✗ ✔

22. Cloning and purification of TER_U1A constructs Cloning I: addition of U1A site Cloning II: addition of HH site Cloning III: mutation of A112G Cloning IV: mutation of U157C U1A GCGC GCGC Hammerhead sequence Constructs ready for in vitro transcription and spontaneous HH cleavage HH cleavage site Purification of U1A and p65 FL

23. Test transcription of TER_U1A00_HH and TER_U1A01_HH TER_U1A00_HH TER_U1A01_HH << DNA template << TER_U1A00_HH (127 nt) << TER_U1A00 (77 nt) << TER_HH (50 nt) 1 U T7 1/10 U T7

24. Refolding of TER_U1A00 and TER_U1A01 Result: No difference in folding pattern. RNAs finally folded in H2O @ 98 deg, 4 min, 4 deg 20 min TER_U1A00 TER_U1A01

25. EMSA of TER_U1A00 and TER_U1A01 with U1A and crystallization trials Crystal screen setup: Nucleix @ RT

26. EMSA of TER_U1A00 and TER_U1A01 with p65 FL and crystallization trials Crystal screen setup: Nucleix, CS1 & 2 @ RT & 4 deg

27. EMSA of TER_U1A00 and TER_U1A01 with U1A and p65 FL: crystallization trials for the ternary complex Crystal screen setup: Nucleix, CS1 & 2 @ RT & 4 deg

28. Future goals NMR: 1.Finish the NMR structure of Cter02dL followed by sHnCter02 (369-519) 2. Work on Cter01dL/pstem4 complex sample Crystallization: 1. Purification of new full length constructs. 2. New TER S1&SL4 constructs. 3. Optimize p65 FL/TER_U1A complex.