RNA modifications stabilize the tertiary structure of tRNAfMet by locally increasing conformational dynamics

Abstract

A plethora of modified nucleotides extends the chemical and conformational space for natural occurring RNAs. tRNAs constitute the class of RNAs with the highest modification rate. The extensive modification modulates their overall stability, the fidelity and efficiency of translation. However, the impact of nucleotide modifications on the local structural dynamics is not well characterized. Here we show that the incorporation of the modified nucleotides in tRNAfMet from Escherichia coli leads to an increase in the local conformational dynamics, ultimately resulting in the stabilization of the overall tertiary structure. Through analysis of the local dynamics by NMR spectroscopic methods we find that, although the overall thermal stability of the tRNA is higher for the modified molecule, the conformational fluctuations on the local level are increased in comparison to an unmodified tRNA. In consequence, the melting of individual base pairs in the unmodified tRNA is determined by high entropic penalties compared to the modified. Further, we find that the modifications lead to a stabilization of long-range interactions harmonizing the stability of the tRNA's secondary and tertiary structure. Our results demonstrate that the increase in chemical space through introduction of modifications enables the population of otherwise inaccessible conformational substates.

Figure 1.

Quantification of modified nucleosides levels in the non-modified (red) and modified (blue) tRNA^fMet constructs. (A) Secondary structure of non-modified and modified tRNA^fMet. (B) LC–MS based quantification of modified nucleosides levels in tRNA^fMet. Both tRNA^fMet constructs were digested to nucleosides and analyzed via LC-MS. The amount of each modification was normalized to the amount of injected RNA molecules. The occurrence of one modified nucleoside per RNA molecule is highlighted with a black line. The modified nucleosides are s⁴U8 (thiouridine 8), D20 (5,6-dihydrouridine 20), C_m32 (2′O-methylcytidine 32), T54 (ribothymidine 54), Ψ55 (pseudouridine 55), and m⁷G46 (7-methylguanosine 46). The measured relative abundance for m⁷G46 is based on the endogenous isoacceptor of tRNA^fMet in E. coli. In E. coli K strains, the initiator tRNA^fMet is encoded by four genes that differ by a single nucleotide at position 46 resulting in two tRNA^fMet species (79,80): metZ, metW and metV encode tRNA^fMet1 with m⁷G46 and metY encode tRNA^fMet2 with A46. Therefore, the cellular tRNA^fMet pool consists of 75% tRNA_G46^fMet1, and 25% of tRNA_A46^fMet2 due to the presence of three genes encoding for m⁷G46 and only one gene encoding for A46 (80,81). In contrast, the results here show only a relative abundance of 27.7% and thus, the second isoacceptor reflecting the tRNA_A46^fMet2 of interest is successfully overexpressed. (C) Chemical structure of analyzed modified nucleosides.

Figure 2.

Selected [¹H,¹H]-strips of a 3D-[¹H,¹⁵N,¹H]-SOFAST-HMQC-NOESY (A), 2D-[¹H,¹⁵N]-BEST-TROSY (B) and 2D-[¹H,¹⁵N]-BEST-TROSY-HNN-COSY experiment (C) of modified tRNA^fMet at 25°C. Secondary structure of the modified tRNA^fMet is shown in (C). Unambiguously assigned imino peaks are highlighted in either purple (acceptor stem), blue (D-arm), yellow (ACSL), or red (TΨC-arm). Experimental details are provided in an extended figure caption in SI.

Figure 3.

2D-[¹H,¹⁵N]-BEST-TROSY experiment of unmodified tRNA^fMet with secondary structure (A) and analysis of chemical shift differences between modified and non-modified tRNA (B). (A) The BEST-TROSY spectrum of the non-modified tRNA^fMet was acquired at 25°C. The secondary structure of the non-modified tRNA^fMet is shown. Unambiguously assigned imino peaks are highlighted in either purple (acceptor stem), blue (D-arm), orange (ACSL) or red (TΨC-arm). (B) Chemical shift difference between the imino resonances of the non-modified and modified tRNA^fMet construct was calculated as absolute values of the difference between corresponding imino signals (Supplementary Table S1 and Supplementary Table S2). Experimental details are provided in an extended figure caption in SI.

Figure 4.

NMR spectroscopic signatures of modified residues. (A) Characterization of N3H3-phosphate group hydrogen bonds through sofast-¹H,³¹P HMQC experiments. The H3 atom of residues U55 (left panel) and Ψ55 (right panel) with the phosphate group of residues A58 is correlated and related to the according HN cross peaks of the corresponding ¹H¹⁵N HSQC, which are given in the panels below. (B) Placement of modified residues within the core region, which are establishing the interaction between D- (pink) and TΨC-loop (blue) as mapped by crystal structure (PDB: 3CW6). (C) The C7H₃ cross peak of T54 detected in ¹H,¹³C HSQC and (D) the methylene-group cross peaks of D20 C5H₂ and C6H₂ in ¹H,¹³C HSQC. The inserts in (C) and (D) reflect the traces along the ¹³C-dimension to highlight the multiplet structure and reveal the respective ¹J_CC couplings. Experimental details are provided in an extended figure caption in SI.

Figure 5.

Temperature coefficients of the proton chemical shifts (A) and CD melting profiles of the non-modified (blue) and modified (grey) tRNA^fMet. (A) Temperature coefficients of the proton chemical shifts for the non-modified tRNA ((¹H), blue bars) and for the modified tRNA^fMet construct ((¹H), red bars). The temperature coefficients were determined through a linear fit of the measured chemical shifts at temperatures ranging from 5 to 45°C (Supplementary Figure S4–S5). (B) CD melting profiles were measured and analyzed as described in the method section. The resulting fitting parameters are shown.

Figure 6.

Comparison of local dynamics and base pair stabilities for modified and unmodified tRNA^fMet. Nucleotide-resolved representation of the L-shaped structure that is color coded to represent the S² order parameter for (A) native tRNA at 25°C and (B) at 40°C; (C) unmodified tRNA at 25°C and (D) at 40°C. The base interactions are annotated according to the nomenclature of Leontis and Westhof (112), modified residues are represented by hexagons instead of circles. The differences in the stabilities of corresponding base pairs () are given for (E) 25°C and (F) 40°C. Positive values shown in blue and green mean that modified tRNA is more stable than unmodified tRNA. In contrast, negative values shown in yellow and red show that unmodified tRNA is more stable than modified tRNA. White highlighted nucleotides represent only a minor difference in between both tRNAs. All data is summarized in Supplementary Tables S9-S12 and S15-S16.