Imino chemical shift assignments of tRNAAsp, tRNAVal and tRNAPhe from Escherichia coli

Abstract

Transfer RNAs (tRNAs) are an essential component of the protein synthesis machinery. In order to accomplish their cellular functions, tRNAs go through a highly controlled biogenesis process leading to the production of correctly folded tRNAs. tRNAs in solution adopt the characteristic L-shape form, a stable tertiary conformation imperative for the cellular stability of tRNAs, their thermotolerance, their interaction with protein and RNA complexes and their activity in the translation process. The introduction of post-transcriptional modifications by modification enzymes, the global conformation of tRNAs, and their cellular stability are highly interconnected. We aim to further investigate this existing link by monitoring the maturation of bacterial tRNAs in E. coli extracts using NMR. Here, we report on the ¹H, ¹⁵N chemical shift assignment of the imino groups and some amino groups of unmodified and modified E. coli tRNA^Asp, tRNA^Val and tRNA^Phe, which are essential for characterizing their maturation process using NMR spectroscopy.

Keywords: Amino groups; Imino groups; NMR; Post-transcriptional modifications; RNA chemical shift assignments; tRNA.

Fig. 1

Chemical structure of common modified residues discussed in the assignment procedures. 4-thiouridine (s⁴U), dihydrouridine (D), 7-methylguanosine (m⁷G), thymidine or 5-methyluridine (T or m⁵U), and pseudouridine (Ψ). Modifications are highlighted in red

Fig. 2

¹H, ¹⁵N chemical shift assignment of the imino and amino groups of E. coli tRNA^Asp(GUC). (a) Sequence and L-shape 2D representation of modified E. coli tRNA^Asp(GUC). Modified residues are represented in red. The nature of interactions between base pairs is described according to the classification by Leontis and Westhof (Leontis and Westhof 2001). (b) (¹H,¹⁵N)-BEST-TROSY spectrum and assignment of the imino groups of unmodified and modified E. coli tRNA^Asp. Signals from the unmodified sample are represented in blue and signals from the modified sample are in red. Asterisks denote peaks in the modified sample that correspond to a subpopulation in which U65 is not modified into Ψ65. (c) (¹H,¹⁵N)-BEST-TROSY-HNN-COSY spectrum showing correlations across hydrogen bonds in the modified tRNA^Asp. (d) (¹H,¹⁵N)-CPMG-NOESY-HSQC enabling the chemical shift assignment of the amino groups in the modified tRNA^Asp. (e) (¹H,¹⁵N)-HSQC spectrum showing the chemical shift assignment of the NH₂-amino groups of modified tRNA^Asp

Fig. 3

¹H,¹⁵N chemical shift assignment of the imino groups of E. coli tRNA^Val(UAC). (a) Sequence and L-shape 2D representation of modified E. coli tRNA^Val(UAC). The same code is used as on Fig. 2. (b) (¹H,¹⁵N)-BEST-TROSY spectrum and assignment of the imino groups of unmodified and modified E. coli tRNA^Val. Signals from the unmodified sample are represented in blue and signals from the modified sample are represented in red. Asterisks denote peaks that correspond to an alternative folding of unmodified tRNA^Val

Fig. 4

¹H,¹⁵N chemical shift assignment of the imino groups of E. coli tRNA^Phe(GAA). (a) Sequence and L-shape 2D representation of modified E. coli tRNA^Phe(GAA). The same code is used as on Fig. 2. (b) (¹H,¹⁵N)-BEST-TROSY spectrum and assignment of the imino groups of unmodified and modified E. coli tRNA^Phe. Signals from the unmodified sample are represented in blue and signals from the modified sample are represented in red. Asterisks denote minor peaks in the unmodified tRNA^Phe corresponding to minor alternative folding conformations

Fig. 5

¹H and ¹⁵N combined chemical shift differences between the imino resonances of the unmodified and modified E. coli tRNAs. Histograms showing the combined chemical shifts difference of assigned nucleotides between unmodified and modified samples of (a) tRNA^Asp(GUC), (b) tRNA^Val(UAC) and (c) tRNA^Phe(GAA)