Nitrogen-15-labeled yeast tRNAPhe: double and two-dimensional heteronuclear NMR of guanosine and uracil ring NH groups

Abstract

5N1-Labeled hypoxanthine and 1,3-15N-labeled uracil were synthesized chemically and used to prepare labeled yeast tRNAPhe biosynthetically. Maps (500 MHz) of 15N chemical shift vs. proton chemical shift were obtained, for each ring NH group, by means of INDOR (difference heterodecoupling) and also by means of a proton-observe two-dimensional method involving coherences of forbidden resonances of the NH system. Resonances of GC11, T54-m1A58, GU4, and A psi 31 were confirmed, assigned, or reassigned. psi 39 was found to be in anti conformation, not syn as previously stated. Almost all the uracil NH group resonances could be separated, but most of the GC resonances are too close even in two dimensions to be separately resolved with the observed 20-Hz 15N line width.

Imino‐proton resonances of yeast tRNAPhe studied by two‐dimensional nuclear Overhauser enhancement spectroscopy

“…To this end, site-specific 15N labeling with heteronuclear decoupling could be helpful [24, 251. Recently, this kind of labeling has been used in another application of 2D NMR to tRNA: i.e. multiple quantum two-dimensional 'H-15N NMR spectroscopy [26,27].…”

NMR and biochemical characterization of recombinant human tRNA3 Lys expressed in Escherichia coli: Identification of posttranscriptional nucleotide modifications required for efficient initiation of HIV-1 reverse transcription

“…Lys are essential for its function as a primer of reverse transcription (Barat et al+, 1991;Isel et al+, 1993Isel et al+, , 1996Lanchy et al+, 1996) (Björk, 1996) and none of these have been described as crucial for priming reverse transcription+ Only m 1 A 58 is important for efficacy and fidelity of (ϩ) strand DNA transfer (Burnett & McHenry, 1997;Auxilien et al+, 1999) (Roy et al+, 1984)+ The methyl of m 7 G 46 (3+86 ppm) resonates at a lower field because of the nearness of the positively charged N7 atom+ It gives a strong NOESY cross-peak to its aromatic H8 proton, which is also shifted downfield at 9+25 ppm (Fig+ 2) and is slowly exchangeable with the solvent, as previously described for m 7 G (Leroy et al+, 1985)+ The methyl at 1+35 ppm corresponds to that of a threonine side chain+ Connectivities within the threonine spin system were confirmed in a TOCSY experiment (not shown)+ The NOE cross-peak between the methyl group of the threonine side chain and its amide proton can be seen in Figure 2+ Finally, the methyl at 2+95 ppm must correspond to the mnm 5 s 2 U 34 nucleotide because the NH at 10+05 ppm has two NOEs with a methyl at 2+95 ppm and a CH 2 group at 3+60 ppm+ The presence of the two dihydrouridines (D) could be detected in the NOESY and the TOCSY experiments, through the observation of correlations between the ring CH 2 methylene groups (not shown)+ These were straightforward to identify, as they resonate between 2+0 and 3+0 ppm, in a region of the spectrum that is otherwise devoid of signal in unmodified RNA+ However, individual assignment of these protons could not be achieved+ Pseudouridines (⌿) contain two imino groups (HN1 and HN3) separated by a CO group+ In an HNCO type experiment performed on a doubly labeled 13 C/ 15 N tRNA 3 Lys , these two imino protons must thus correlate to the same intervening carbonyl+ We were able to detect such a double correlation for ⌿ 55 (data not shown)+ For ⌿ 39 , only the HN1 imino proton was observed, presumably because the other imino group is in a fast exchange rate with solvent+ Confirmation of the ⌿ assignments came from 1 H{ 15 N} HMQC experiment (Fig+ 2B) in which both N1 imino groups of ⌿ 39 and ⌿ 55 resonate, respectively, at 133 ppm and 136 ppm in the nitrogen dimension, quite far from both the N3 iminos of Us (159-163 ppm) and the N1 iminos of Gs (146-151 ppm)+ Furthermore, the HNCO experiment also revealed the correlation originating from the threonine moiety of the t 6 A 37 nucleotide, which is traditionally observed across an amide bond+ As sulphur nuclei are not readily amenable to NMR studies, the presence of thiolated nucleotides was investigated by looking for their sensitivity to chemical Lys oxidation+ Iodine treatment of tRNA selectively promotes the formation of disulphide bridges between 2-thio-uridine residues (Carbon et al+, 1965)+ This, in turn, inactivates the tRNA, which forms covalent dimers+ When applied to our recombinant tRNA, this treatment induces a dimerization of a fraction of the molecules (15-30%), as observed by denaturing gel electrophoresis (not shown)+ This indicates that a significant fraction of the recombinant tRNA contains thiolated nucleotides+ This must correspond to s 2 U 34 , because we checked that s 4 U 8 is not reactive to iodine under the same co...…”

Initial analysis of 750 MHz NMR spectra of selectively 15N‐G,U labelled E. coli 5S rRNA

“…This experiment allows clear differentiation between guanine and uridine imino groups on the basis of the characteristic 15N chemical shifts [33]. A further, less stringent, indication for the type of base pairs is given by the 'H chemical shifts.…”

“…50-60 Hz) and 15N (approx. [30][31][32][33][34][35] resonances as well as to the rather facile exchange of such labile hydrogens with solvent water [35]. For this purpose we have developed a modified 3D 15N-edited NOESY experiment (Fig.…”