Computational protocol: Effect of pseudouridylation on the structure and activity of the catalytically essential P6.1 hairpin in human telomerase RNA

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[…] NMR spectra were recorded on Bruker DRX or Avance 500, 600 and 800 MHz spectrometers equipped with HCN cryoprobes or QXI probe. For the peak assignments, exchangeable proton spectra were taken in 95% H2O/5% D2O at 283 K and non-exchangeable proton spectra in 99.996% D2O (Sigma) at 293 K. NMR spectra were processed and analyzed using XWINNMR 3.5 (Bruker) and Sparky 3.110. (University of California, San Francisco, CA, USA.) The standard H1′-base proton sequential assignments were initially obtained from analysis of 2D NOESY and 2D TOCSY experiments on unlabeled RNA samples. The assignments for all non-exchangeable protons were achieved from analysis of 2D NOESY, 2D 1H-13C HSQC, 2D HCCH-COSY and 3D HCCH-TOCSY (,). A suite of 2D-filtered/edited proton NOESY (F2f, F1fF2e, F1fF2f and F1eF2e) experiments on 13C,15N-A,C,G-labeled Ψ4-P6.1 RNA were used to resolve ambiguous assignments in overlapped regions and obtain NOE restraints, as previously described (). The imino and amino exchangeable protons were assigned from 2D NOESY, 2D 15N-correlated CPMG NOESY and 2D 1H-15N HMQC spectra acquired on samples in 95% H2O/5% D2O. The 2′-hydroxyl protons were identified by scalar coupling to H2′ protons observed in 2D DIPSI TOCSY (τm = 43 ms) () and by NOE correlation with H1′s observed using 2D flip-back water gate NOESY (τm = 50 ms) (40,41; Supplementary Figure S2) Residual dipolar couplings (RDCs) for Ψ4-P6.1 RNA were measured from the differences in the splitting in the 1H dimension of HSQC spectra () (800 MHz), in the absence and presence of Pf1 phage (16 mg/ml, ASLA Biotech, Ltd).Interproton distances were generated from the 2D NOESY spectrum (τm = 100 ms) as well as 2D-filtered/edited NOESY spectra (). NOE distance restraints were classified as strong (1.8–3.5Å), medium (1.8–4.5Å), weak (1.8–5.5Å) and very weak (1.8–6.5Å). A total of 137 intra-nucleotide and 136 inter-nucleotide NOE derived distance restraints and 88 dihedral angle restraints (α, β, γ, δ, ε, χ, ν2 and ζ) were used in the structure calculations, where the δ, ε and χ were determined experimentally as described below. The α, β, γ, ν2 and ζ dihedral angles for the nucleotides involved in base pairs in the helix were constrained to the A-form values (−62.1 ± 30°, 180.1 ± 30°, 47.4 ± 30°, 37.3 ± 30° and −74.7 ± 30°, respectively). 3JH2′P and 3JCP were measured using 31P spin echo difference CT HSQCs to determine the ε dihedral angles for the loop residues (). Final structure calculations included hydrogen bond distance restraints for the five Watson–Crick base pairs, and a non-Watson–Crick base pair as previously described (). Weak base pair planarity and hydrogen bonding restraints were used for unambiguously assigned base pairs. Nucleotide G309, which showed a strong intra-nucleotide H1′-aromatic NOE and a weak intra-nucleotide H2′-aromatic NOE in a 2D NOESY (τm = 50 ms), was restrained to the syn glycosidic torsional angle (χ = 60 ± 30°), while all other nucleotides were restrained to ‘anti’ (χ = −160 ± 30°). The ribose conformation was constrained to C2′-endo (δ = 145 ± 30°) for residues G308 and G309, based on a strong NOE cross peak between the H1′ and H2′ protons and observable H1′-H3′ couplings in a 50-ms mixing time 2D TOCSY spectrum (). A total of 27 one bond C-H RDCs (1DC1′H1′, 1DC8H8, 1DC6H6, 1DC5H5 and 1DC2H2) were obtained. One hundred initial structures of Ψ4-P6.1 were calculated using XPLOR-NIH 2.9.8 starting from an extended, unfolded RNA using the NOE distance and dihedral angle restraints following standard XPLOR protocols. The 25 lowest energy structures were further refined by inclusion of RDCs for the final structures. Experimental restraints and structural statistics for the 20 lowest energy structures are shown in . All structures were viewed and analyzed using MOLMOL () and PYMOL (DeLano Scientific LLC). […]

Pipeline specifications

Software tools Sparky, Xplor-NIH, MOLMOL, PyMOL
Applications NMR-based proteomics analysis, Protein structure analysis
Organisms Homo sapiens
Chemicals Pseudouridine