1. Tetrahymena telomerase Nhp2p Chin Ju Park January 22, 2008 Progress Reports 6

2. Tetrahymena telomerase ribonucleoprotein complex functions as a reverse transcriptase enzyme adds telomeric repeats to chromosome 3’ ends TEN (1-195)RBD (195-516)

3. tTERT(195-516) with TBE RBD domain (195-516) 20mM MES, 250mM NaCl, 0.4mM DTT pH 6.0, 277K, 800MHz Precipitation occurred when RBD and RNA were mixed together at pH 5~7. (even in dilute concentration ~10 uM. Mixing order and different kind of salt doesn’t make any improvement)

4. tTERT(195-516):tTER(1-59)=1:2 (protein conc. ~0.1mM) 20mM Tris, 100mM NaCl, 1mM MgCl 2, 1mM DTT, pH 8.0, 280K Mulder, TROSY (# Protein only : no signals at pH 8.0)

5. NGKATSNNNQNNANLSNEKKQENQYIYPEIQRSQIFYCNHMGREPGVFKSSFFNYSEIKKGFQFKVIQEKLQGRQFINSDKIKP DHPQTIIKKTLLKEYQSKNFSCQEERDLFLEFTEKIVQNFHNINFNYLLKKFCKLPENYQSLKSQVKQIVQSENKANQQSCENLF NSLYDTEISYKQITNFLRQIIQNCVPNQLLGKKNFKVFLEKLYEFVQMKRFENQKVLDYICFMDVFDVEWFVDLKNQKFTQKRK YISDKRKILGDLIVFIINKIVIPVLRYNFYITEKHKEGSQIFYYRKPIWKLVSKLTIVKLEEENLEKVE EKL 1020304050607080 90100110 120130 140 150160 170 190180200 210 220 230 240 250 260290 270 280 300 310 320 Restricted Proteolysis (Trypsin, 37  C, 20 min) Not consistent with 2ndary structure prediction Unstructured N-term is known to be crucial for RNA counterpart binding CD Data proves existence of 2ndary structures but lack of evidence for 3rdary structure. # RBD of telomerase structure was Deposited to PDB (2r4g) : 254-519 *CP2-motif is not included

6. Structure of the RNA-Binding Domain of Telomerase: Implications for RNA Recognition and Binding Structure 15 1403-1412 (2007) More flexible T-motif CP-motif 12 alpha helices, 2 beta sheets Two asymmetric halves ~120° QFP-motif

7. T-motif: narrow, well-defined Pocket CP-motif: shallow, wide, Highly positively charged Cavity Close each other, forming a Single large, positively Charged surface area

8. Mutated residues that affect RNA binding and telomerase activity are boxed. T- and CP- motif 1)F476A, Y477A, T479A, E480A, R492A, W496A in T-motif : severe loss of RNA binding affinity and telomerase activity 2) L327A, K329A, C331A, P334A in CP-motif: moderate loss of RNA binding affinity And telomerase activity * L327, C331 - F476, R492

9. TRE N terminal of RBD is necessary for RNA binding T-pocket is too narrow (W496 and Y477), cannot accommodate a nucleotide base without conformational change. ssRNA binding? TBE? CP-motif is more wider: interact With dsRNA (stem I or II?) CP-2 : 226-237

10. Work in Progress 254-519 was cloned into pET46 (N-terminal his tag) vector. 15 N HSQC RNA binding test

11. Facts of Nhp2p and hTR hTR has H/ACA domain in its 3’ half. Mol. Cell 1999 19 567-576 The loop of the terminal stem (cR7) is required for hTR accumulation. Mol. Cell 2003 11 1361-1372 Stable hTR expression in yeast requires Cbf5p, Nhp2p, and Nop10p. Yeast telomerase RNA doesn’t have H/ACA domain and doesn’t bind to those proteins. Nucleic Acids Res. 2001 29 598-603 Nhp2p binds irregular stem-loop structures of RNA : tested with yeast snoRNA (snR36). Nucleic Acids Res.2001 29 2733-2746 Archaeal L7Ae and eukaryal Snu13p(yeast), 15.5K (human) recognize K-turn RNA Motif. J. Mol. Biol. 2004 342 757-773 Crystal Structure of Archaeal H/ACA RNP particles : L7Ae contacts with terminal K-turn and Nop10 Nature 2006 443 302-307

12. Truncation Mutation P→A Homology modeling N-ter Homology Modeling template : yeast RNA splicing factor snu13p (2ALE.pdb) Sequence identity: 38% CPH model (http://www.cbs.dtu.dk) Yeast Nhp2 protein : Structure prediction α1α1 α2α2 β1β1 β2β2 α3α3 α4α4

13. Previous Results 20mM HEPES-KOH, 200mM KCl, pH 7.4, 300K Nhp2p(44-176)_P103A Backbone assignment : 70% done Residues near prolines are not shown in triple resonance experiments. Arg/Glu buffers are not helpful

14. U21 U5 G4 G420 G419 G6 G2 Nhp2p:cR7t=1:1.2 cR7t only cR7t with Nhp2p(44-176)_P103A 20mM HEPES-KOH, 200mM KCl, pH 7.4, 280K, 500MHz

15. Blue: Nhp2p only Red: Nhp2p:cR7t = 1:0.6 Green: Nhp2p:cR7t = 1:1.8 Nhp2p _P103A, 20mM HEPES-KOH, 200mM KCl, pH 7.4, 300K, 500MHz

16. Interaction with Nop10p Interaction with K-turn RNA

17. Nhp2p with Nop10p 1:0 1:0.5 1:1 1:2

18. Nhp2p with Nop10p 1:3 Nhp2p _P103A, 20mM HEPES-KOH, 200mM KCl, pH 7.4, 300K, 500MHz A B A B S106 A8 No consistency with L7Ae-Nop10 interaction

19. RNA Preparation: Archeal H/ACA RNA

20. Red: Nhp2p only Green: Nhp2p:Ar RNA = 1:0.5 Blue: Nhp2p:Ar RNA = 1:1 Nhp2p with Ar H/ACA RNA Almost no chemical shift perturbations Some signals disappeared Proteins were precipitated with direct addition of RNA Dilution→ Mix→ Concentration Still invisible aggregation Peaks are not recovered until 1:3 Nhp2p _P103A, 20mM HEPES-KOH, 200mM KCl, pH 7.4, 300K, 500MHz

21. RNA Preparation: cR7 wild type U7 G21

22. Nhp2p with cR7 wt Nhp2p _P103A, 100mM NaPi, 100mM NaCl, pH 6, 293K, 500MHz Blue: Nhp2p Only Magenta: 1:1 Even mixing in dilute condition makes white precipitation instantly Only few signal remains in random coil region

23. Nhp2p in different pH Blue: 100mM NaPi, 100mM NaCl, pH 6 Pink: 20mM HEPES, 200mM KCl, pH 7.4 New peaks are appeared in pH 6, especially for G/T/S * 15.5K solution structure (Biochemistry 2007 46 14979-14986)

24. Progress in assignments MPAVLPFAKP LASKKLNKKV LKTVKKASKA KNVKRGVKEV VKALRKGEKG LVVIAGDISA ADVISHIPVL CEDHSVPYIF IPSKQDLGAA GATKRPTSVV FIVPGSNKKK DGKNKEEEYK ESFNEVVKEV QAL ~85% assignments *underlined residues are not assigned

25. Chemical shift index The Chemical shift index (CSI) is a commonly accepted procedure to establish the secondary structure of proteins based on chemical shift differences with respect to some predefined 'random coil' values. It can be applied from the measured HA, CA, CB and CO chemical shifts for each residue in a protein. MPAVLPFAKP LAS KKLNKKV LKTVKKAS KA KNVKRG VKEV VKALRKGEKG LVVIAGDISA ADVISHIPVL CEDHSVPYIF IPSKQDLGAA GATKRPTSVV FIVPGSNKKK DGKNKEEEYK ESFNEVVKEV QAL If measured chemical shifts for CA/CO are greater than random value, +1 Alpha helix is defined when four or more "1" CA/CO are sequentially found. MPAVLPFAKP LAS KKLNKKV LKTVKKAS KA KNVKRG VKEV VKALRKGEKG LVVIAGDISA ADVISHIPVL CEDHSVPYIF I PSKQDLGAA GATKRPTSVV FIVPGSNKKK DGKNKEEEYK ESFNEVVKEV QAL Secondary structure prediction based on sequence homology *Alpha3 is not established (P)

27. Works in Progress Assigned spectra CBCACONH, HNCACB HNCO, HN(CA)CO Need to assign HCC(CO)NH 15 N NOESY-HSQC (in progress) Need to take 13 C NOESY-HSQC CC(CO)NH Dihedral angle restraints from TALOS Peak picking, chemical shift lists CYANA calculation Need to find condition for RNA interaction at lower pH

28. U1A purification with Nakkyoon p65-NCL partial backbone assignment with Mahavir ETC.