NMR investigation of the effect of selective modifications in the anticodon loop on the conformation of yeast transfer RNAPhe

doi:10.1016/0005-2787(75)90227-0

Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis

Volume 395, Issue 1, 2 June 1975, Pages 1-4

https://doi.org/10.1016/0005-2787(75)90227-0 Get rights and content

Abstract

Changes in the 300 MHz proton NMR spectrum of yeast tRNA^Phe induced by the removal of the Y-base from the anticodon loop are reversed when the dye proflavine is incorporated in its place. These observations correlate with our earlier interpretation of the NMR data that removal of the Y-base causes a change in the conformation of the anticodon stem. Such changes in stem conformation may in part be responsible for the differences in the biochemical properties of tRNA^Phe, tRNA^Phe_PF and tRNA^Phe_−Y.

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Cited by (18)

Probing the conformation of human tRNA<inf>3</inf>Lys in solution by NMR
2007, FEBS Letters
Human ${tRNA}_{3}^{Lys}$ acts as a primer for the reverse transcription of human immunodeficiency virus genomic RNA. To form an initiation complex with genomic RNA, ${tRNA}_{3}^{Lys}$ must reorganize its secondary structure. To provide a starting point for mechanistic studies of the formation of the initiation complex, we here present solution NMR investigations of human ${tRNA}_{3}^{Lys}$ . We use a straightforward set of NMR experiments to show that ${tRNA}_{3}^{Lys}$ adopts a standard transfer ribonucleic acid tertiary structure in solution, and that Mg²⁺ is required for this folding. The results underscore the power of NMR to reveal rapidly the conformation of RNAs.
Naturally-occurring Modification Restricts the Anticodon Domain Conformational Space of tRNAPhe
2003, Journal of Molecular Biology
Citation Excerpt :
Very early model building studies of tRNA suggested that the conformational freedom of the single-stranded anticodon loop was restricted significantly, and that alkylation of purine 37 may enhance hydrophobic stabilization of a 3′-stacked conformation.61 Later NMR studies of the native and proflavine modified position 37 also indicated that the hydrophobic nature of the nucleoside was important to anticodon loop structure.62 However, studies of yeast tRNAPhe in which the inherent fluorescence of the tricyclic yW37 modification at position 37 was monitored, reported that the base was not always stacked.63
Post-transcriptional modifications contribute chemistry and structure to RNAs. Modifications of tRNA at nucleoside 37, 3′-adjacent to the anticodon, are particularly interesting because they facilitate codon recognition and negate translational frame-shifting. To assess if the functional contribution of a position 37-modified nucleoside defines a specific structure or restricts conformational flexibility, structures of the yeast tRNA^Phe anticodon stem and loop (ASL^Phe) with naturally occurring modified nucleosides differing only at position 37, ASL^Phe-(Cm₃₂,Gm₃₄,m⁵C₄₀), and ASL^Phe-(Cm₃₂,Gm₃₄,m¹G₃₇,m⁵C₄₀), were determined by NMR spectroscopy and restrained molecular dynamics. The ASL structures had similarly resolved stems (RMSD∼0.6 Å) of five canonical base-pairs in standard A-form RNA. The “NOE walk” was evident on the 5′ and 3′ sides of the stems of both RNAs, and extended to the adjacent loop nucleosides. The NOESY cross-peaks involving U₃₃ H2′ and characteristic of tRNA's anticodon domain U-turn were present but weak, whereas those involving the U₃₃ H1′ proton were absent from the spectra of both ASLs. However, ASL^Phe-(Cm₃₂,Gm₃₄,m¹G₃₇,m⁵C₄₀) exhibited the downfield shifted ³¹P resonance of U₃₃pGm₃₄ indicative of U-turns; ASL^Phe-(Cm₃₂,Gm₃₄,m⁵C₄₀) did not. An unusual “backwards” NOE between Gm₃₄ and A₃₅ (Gm₃₄/H8 to A₃₅/H1′) was observed in both molecules. The RNAs exhibited a protonated A⁺₃₈ resulting in the final structures having C₃₂·A⁺₃₈ intra-loop base-pairs, with that of ASL^Phe-(Cm₃₂,Gm₃₄,m¹G₃₇,m⁵C₄₀) being especially well defined. A single family of low-energy structures of ASL^Phe-(Cm₃₂,Gm₃₄, m¹G₃₇,m⁵C₄₀) (loop RMSD 0.98 Å) exhibited a significantly restricted conformational space for the anticodon loop in comparison to that of ASL^Phe-(Cm₃₂,Gm₃₄,m⁵C₄₀) (loop RMSD 2.58 Å). In addition, the ASL^Phe-(Cm₃₂,Gm₃₄,m¹G₃₇,m⁵C₄₀) average structure had a greater degree of similarity to that of the yeast tRNA^Phe crystal structure. A comparison of the resulting structures indicates that modification of position 37 affects the accuracy of decoding and the maintenance of the mRNA reading frame by restricting anticodon loop conformational space.
The Use of Nuclear Magnetic Resonance in the Study of Transfer RNA Structure in Solution
1979, Methods in Enzymology
The chapter discuses the use of nuclear magnetic resonance (NMR) in the study of transfer RNA structure in solution. At both extremes of the proton NMR range there are a manageable number of usually resolved resonances, and it is from these regions of the spectrum that the most detailed, interpretable information on tRNA structure in solution is obtained. The high-field region (between 0 and -4 ppm from DSS) contains aliphatic protons from modified bases, for example, the methyl resonances of methylated nucleotides or the methylene resonances of dihydrouridine. These are nonexchangeable; therefore, they can be studied in either H₂O or DzO solutions. The low-field region (between -11 and -15 ppm) contains the imino ring NH protons, which are involved in the hydrogen bonding of complementary base pairs. This region of the spectrum is extremely informative in that each base pair contains only one ring NH hydrogen bond; therefore the number of low- field resonances directly reveals the number of stable base pairs in solution. Low-field proton spectroscopy cannot be carried out in D₂O solutions because of the solvent exchange of the hydrogen-bonded ring NH protons. For the same reason, the rapid exchange of unpaired ring NH protons in contact with water effectively eliminates these protons from the low-field spectrum by time averaging into the H₂O peak at ca. -4.7ppm. A distinct disadvantage of this form of NMR spectroscopy is the presence of the large (110 M) H₂O proton peak; however, this is amply compensated by the ability to monitor a single natural reporter group from each base pair in a tRNA molecule.
Minor components in transfer RNA: The location-function relationships
1978, Progress in Biophysics and Molecular Biology
The paper revises data concerning the relationship between the structure, location and function of minor components in tRNA. It draws attention to some formerly ignored features in the regular distribution of minor components in tRNA, notably in D and extra loops. An analysis of data in literature suggests a hypothesis explaining the function of a series of minor components of tRNA, especially of modified and hypermodified bases next to the 3′-end of anticodon, and the minor components in D and extra loops. It is postulated that at least some minor components participate in the covalent fixation of tRNA on ribosome. The formation of methylene cross-links from tRNA methyl groups appears the most probable mechanism of this fixation. Some known biochemical and chemical reactions, cited in the paper, could serve as models of the reversible conversion of tRNA methyl to methylene groups.
High-Resolution Nuclear Magnetic Resonance Investigations of the Structure of tRNA in Solution
1976, Progress in Nucleic Acid Research and Molecular Biology
This chapter reviews that high-resolution NMR has been used to provide information on the secondary and tertiary structure of tRNA molecules in solution. It focuses on low field resonances from the ring nitrogen protons involved in base pairing, as these have provided most of the information to date. NMR complements and corroborates the recent X-ray diffraction studies on one particular tRNA, yeast tRNA^Phe, and permits the crystal results on this one tRNA to be generalized to an entire class of tRNA. In this way, the NMR results have served as a bridge to justify the extension of the crystallographic results to tRNA^Phe and other tRNAs in solution. The chapter also discusses that NMR can be used to treat tRNA structure and function problems that are currently unapproachable by X-ray diffraction analysis. NMR has been used to work out plausible structures for the denatured conformers of two tRNAs, and for the dimer of another. The alteration of tRNA conformation as a result of several different chemical modifications removal or replacement of bases in the anticodon loop has also been analyzed. NMR experiments have provided interesting insight into the interaction of tRNA molecules with intercalating drugs and dyes, which may find application in other biochemical and physical chemical studies of tRNA protein interactions.
Long-range conformational trasition in yeast tRNAPhe, induced by the Y-base removal and detected by chloroacetaldehyde modification
1983, Nucleic Acids Research

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Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis

Abstract

References (8)

J. Mol. Biol.

Biochim. Biophys. Acta

FEBS Lett.

Accts. Chem. Res.

Cited by (18)

Probing the conformation of human tRNA<inf>3</inf><sup>Lys</sup> in solution by NMR

Naturally-occurring Modification Restricts the Anticodon Domain Conformational Space of tRNA<sup>Phe</sup>

The Use of Nuclear Magnetic Resonance in the Study of Transfer RNA Structure in Solution

Minor components in transfer RNA: The location-function relationships

High-Resolution Nuclear Magnetic Resonance Investigations of the Structure of tRNA in Solution

Long-range conformational trasition in yeast tRNA<sup>Phe</sup>, induced by the Y-base removal and detected by chloroacetaldehyde modification