Complete nuclear magnetic resonance signal assignments and initial structural studies of [13C]methyl-enriched yeast transfer ribonucleic acid

Agris, Paul F.; Kovacs, Shirley A. H.; Smith, Christine E.; Kopper, Randall A.; Schmidt, Paul G.

doi:10.1021/bi00275a013

Cited by 19 publications

(5 citation statements)

References 25 publications

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“…Methylation of A-58 and G-46 to m1A and m7G, respectively, in tRNA would normally produce ring structures labile to some degree under physiological conditions, but primarily under alkaline conditions (>pH 8). Now we are able to solve problems mentioned in a paper (11), whose authors were expecting the m7G residue of tRNA to be deprotonated in position N(1) under physiological conditions and in our own observations (32,33) of unexplained carbon chemical shift changes for m1A and m7G in tRNA. We know now that protonated forms of m1A and m7G are present in native, ter-tiary structure tRNA, by virtue of tertiary structure hydrogen bonds, and that neutral forms are present only when the tRNA molecule is thermally denatured (Table 1) or when Mg2 ions are removed (21).…”

Section: Discussionmentioning

confidence: 74%

Comparative structural analysis of 1-methyladenosine, 7-methylguanosine, ethenoadenosine and their protonated salts IV:¹H,¹³C, and¹⁵N NMR studies at natural isotope abundance

1986

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The 1H, 13C, and 15N NMR spectra of neutral and protonated forms of the nucleosides 1-methyladenosine (m1A), 7-methylguanosine (m7G) and ethenoadenosine (EA), as a model compound, have been analyzed in order to assign the site of protonation in m1A and m7G. Protonation of these nucleosides occurs in the pyrimidine ring of m1A and EA and in the imidazole ring of m7G, with the charge being distributed rather than localized. Structural differences for both m1A and m7G were observed in solution and compared with those existing in the crystal state of monomers as well as in tRNA where these nucleosides occur quite often. The protonated nucleoside structures in solution compared favorably in sugar pucker and glycosidic bond conformations with x-ray crystallographic data. Methyl group carbon chemical shifts of the protonated mononucleosides corresponded to those of the methyls of the respective nucleosides in native tRNA structures. Therefore, the tRNA methyl group carbon chemical shifts are indicative of fully protonated nucleosides in the native, three dimensional structure of the nucleic acid.

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Section: Discussionmentioning

confidence: 74%

Comparative structural analysis of 1-methyladenosine, 7-methylguanosine, ethenoadenosine and their protonated salts IV:¹H,¹³C, and¹⁵N NMR studies at natural isotope abundance

1986

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“…in which the naturally occurring methyl groups had been 13C enriched in vivo (Agris et al, 1983). The good results achieved from the carbon NMR of unfractionated tRNA demonstrated the potential of studying a purified single [13C]methyl-enriched tRNA species.…”

Section: Resultsmentioning

confidence: 99%

“…Thus, a variety of specific environs of known biological importance (Bjork, 1984) would be probed by such methyl groups in the molecule with the use of 13C NMR. In order to enrich the methyl groups of yeast tRNA for this isotope, a methionine auxotroph has been utilized for in vivo incorporation of [13C]methylmethionine (Agris et al, 1983). Transfer RNA extracted from such a culture demonstrated 13C-enriched NMR signals in relatively short accumulation times.…”

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confidence: 99%

Nuclear magnetic resonance signal assignments of purified [13C]methyl-enriched yeast phenylalanine transfer ribonucleic acid

Smith¹,

Schmidt²,

Petsch³

et al. 1985

Biochemistry

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Yeast tRNA Phe, enriched in carbon-13 specifically at the naturally occurring methyl groups, has been produced through biosynthesis, then purified, and analyzed. Transfer RNA Phe was purified from the [13C]methyl-enriched, unfractionated tRNA that had been extracted from a methionine auxotroph of Saccharomyces cerevisiae [Agris, P. F., Kovacs, S. A. H., Smith, C., Kopper, R. H., & Schmidt, P. G. (1983) Biochemistry 22, 1402-1408]. The yeast had been grown in minimal medium supplemented with [13C]methylmethionine. Transfer RNA Phe purity and the full extent of nucleoside modification were confirmed by high-performance liquid chromatography of constituent nucleosides with simultaneous UV spectral identification and quantitation. Mass spectometry of [13C]methyl-enriched nucleosides and NMR of the tRNA indicated an enrichment of at least 70 atom %. Twelve resolved and prominent carbon-13 NMR signals from the tRNA were seen between 10 and 60 ppm. These have been assigned to 13 of the 14 naturally occurring methyl groups. However, the partially resolved signals assigned to the two 5-methylcytidines could not be assigned to their specific nucleoside positions of either 40 or 49 in the molecule. In addition, the partially resolved signals of the two methyl esters of wybutosine could not be distinguished. The methyl group found not to be enriched with 13C is bound to the ring carbon in the hypermodified nucleoside wybutosine (Y). A 13th enriched signal downfield (120.9 ppm) has been assigned to one of the two carbons added to guanosine to form the third ring in the biosynthesis of Y. The 13C enrichment of this ring carbon demonstrates its origin from the methionine methyl group.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…Materials. Transfer RNAphe was isolated from [methyl-13C] methionine-fed cultures of S. cerevisae met-trp-(RH762) and purified as previously described (Agris et al, 1983;Smith et al, 1985).…”

Section: Methodsmentioning

confidence: 99%

“…Transfer RNA from yeast or Escherichia coli can be enriched in 13C at selected loci by using [m<?r/ry/-13C] methionine for the incorporation in vivo of [13C] methyl groups during synthesis of modified nucleosides (Agris et al, 1975(Agris et al, , 1983Tompson et al, 1979). This technique permits sufficient signal-to-noise ratios to be obtained with the necessarily limited tRNA quantities, and it provides a sound starting point for peak assignments.…”

Section: -5131]mentioning

confidence: 99%

Internal motions in yeast phenylalanine transfer RNA from carbon-13 NMR relaxation rates of modified base methyl groups: a model-free approach

Schmidt

Sierzputowska-Gracz²,

Agris³

1987

Biochemistry

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Internal motions at specific locations through yeast phenylalanine tRNA were measured by using nucleic acid biosynthetically enriched in 13C at modified base methyl groups. Carbon NMR spectra of isotopically enriched tRNA(Phe) reveal 12 individual peaks for 13 of the 14 methyl groups known to be present. The two methyls of N2,N2-dimethylguanosine (m22G-26) have indistinguishable resonances, whereas the fourteenth methyl bound to ring carbon-11 of the hypermodified nucleoside 3' adjacent to the anticodon, wyosine (Y-37), does not come from the [methyl-13C]methionine substrate. Assignments to individual nucleosides within the tRNA were made on the basis of chemical shifts of the mononucleosides [Agris, P. F., Kovacs, S. A. H., Smith, C., Kopper, R. A., & Schmidt, P. G. (1983) Biochemistry 22, 1402-1408; Smith, C., Schmidt, P. G., Petsch, J., & Agris, P. F. (1985) Biochemistry 24, 1434-1440] and correlation of 13C resonances with proton NMR chemical shifts via two-dimensional heteronuclear proton-carbon correlation spectroscopy [Agris, P. F., Sierzputowska-Gracz, H., & Smith, C. (1986) Biochemistry 25, 5126-5131]. Values of 13C longitudinal relaxation (T1) and the nuclear Overhauser enhancements (NOE) were determined at 22.5, 75.5, and 118 MHz for tRNA(Phe) in a physiological buffer solution with 10 mM MgCl2, at 22 degrees C. These data were used to extract two physical parameters that define the system with regard to fast internal motion: the generalized order parameters (S2) and effective correlation times (tau e) for internal motion of the C-H internuclear vectors.(ABSTRACT TRUNCATED AT 250 WORDS)

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Complete nuclear magnetic resonance signal assignments and initial structural studies of [13C]methyl-enriched yeast transfer ribonucleic acid

Cited by 19 publications

References 25 publications

Comparative structural analysis of 1-methyladenosine, 7-methylguanosine, ethenoadenosine and their protonated salts IV:¹H,¹³C, and¹⁵N NMR studies at natural isotope abundance

Comparative structural analysis of 1-methyladenosine, 7-methylguanosine, ethenoadenosine and their protonated salts IV:¹H,¹³C, and¹⁵N NMR studies at natural isotope abundance

Nuclear magnetic resonance signal assignments of purified [13C]methyl-enriched yeast phenylalanine transfer ribonucleic acid

Internal motions in yeast phenylalanine transfer RNA from carbon-13 NMR relaxation rates of modified base methyl groups: a model-free approach

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Complete nuclear magnetic resonance signal assignments and initial structural studies of [13C]methyl-enriched yeast transfer ribonucleic acid

Cited by 19 publications

References 25 publications

Comparative structural analysis of 1-methyladenosine, 7-methylguanosine, ethenoadenosine and their protonated salts IV:1H,13C, and15N NMR studies at natural isotope abundance

Comparative structural analysis of 1-methyladenosine, 7-methylguanosine, ethenoadenosine and their protonated salts IV:1H,13C, and15N NMR studies at natural isotope abundance

Nuclear magnetic resonance signal assignments of purified [13C]methyl-enriched yeast phenylalanine transfer ribonucleic acid

Internal motions in yeast phenylalanine transfer RNA from carbon-13 NMR relaxation rates of modified base methyl groups: a model-free approach

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Comparative structural analysis of 1-methyladenosine, 7-methylguanosine, ethenoadenosine and their protonated salts IV:¹H,¹³C, and¹⁵N NMR studies at natural isotope abundance

Comparative structural analysis of 1-methyladenosine, 7-methylguanosine, ethenoadenosine and their protonated salts IV:¹H,¹³C, and¹⁵N NMR studies at natural isotope abundance