The human mitochondrial tRNAMet: structure/function relationship of a unique modification in the decoding of unconventional codons

Abstract

Human mitochondrial mRNAs utilize the universal AUG and the unconventional isoleucine AUA codons for methionine. In contrast to translation in the cytoplasm, human mitochondria use one tRNA, hmtRNA(Met)(CAU), to read AUG and AUA codons at both the peptidyl- (P-), and aminoacyl- (A-) sites of the ribosome. The hmtRNA(Met)(CAU) has a unique post-transcriptional modification, 5-formylcytidine, at the wobble position 34 (f(5)C(34)), and a cytidine substituting for the invariant uridine at position 33 of the canonical U-turn in tRNAs. The structure of the tRNA anticodon stem and loop domain (hmtASL(Met)(CAU)), determined by NMR restrained molecular modeling, revealed how the f(5)C(34) modification facilitates the decoding of AUA at the P- and the A-sites. The f(5)C(34) defined a reduced conformational space for the nucleoside, in what appears to have restricted the conformational dynamics of the anticodon bases of the modified hmtASL(Met)(CAU). The hmtASL(Met)(CAU) exhibited a C-turn conformation that has some characteristics of the U-turn motif. Codon binding studies with both Escherichia coli and bovine mitochondrial ribosomes revealed that the f(5)C(34) facilitates AUA binding in the A-site and suggested that the modification favorably alters the ASL binding kinetics. Mitochondrial translation by many organisms, including humans, sometimes initiates with the universal isoleucine codons AUU and AUC. The f(5)C(34) enabled P-site codon binding to these normally isoleucine codons. Thus, the physicochemical properties of this one modification, f(5)C(34), expand codon recognition from the traditional AUG to the non-traditional, synonymous codons AUU and AUC as well as AUA, in the reassignment of universal codons in the mitochondria.

Figure 1

Primary sequence and secondary structure of the human mtRNA^Met_CAU and its anticodon stem and loop domain (hmtASL^Met_CAU-Ψ₂₇;f⁵C₃₄). Primary sequence and secondary structure of the human (a) mtRNA^Met_CAU and the (b) hmtASL^Met_CAU-Ψ₂₇;f⁵C₃₄. (c) Chemical structure of the 5-formylcytydine modification, f⁵C₃₄. Numbering of the nucleobase atoms is shown. (d) Nucleoside sequence and secondary structure of the anticodon stem and loop domains of the yeast tRNA^Met_i (left) and E.coli tRNA^Met_m (right).

Figure 2

¹H and ³¹P NMR spectra (1D) of the hmtASL^Met_CAU. (a) ¹H NMR spectrum of the hmtASL^Met_CAU-Ψ₂₇;f⁵C₃₄. The H₂O-exchangeable proton resonances, as well as the formyl-proton (H7) resonance of f⁵C₃₄ are shown. (b) ³¹P-NMR spectra (1D) of the hmtASL^Met_CAU-Ψ₂₇ (top) and the hmtASL^Met_CAU-Ψ₂₇;f⁵C₃₄ (bottom). The ³¹P resonances for f⁵C₃₄, C₃₄, A₃₇ and U₃₆ are denoted in red.

Figure 3

The hmtASL^Met_CAU NOSEY spectra. NOESY spectra of hmtASL^Met_CAU-Ψ₂₇;f⁵C₃₄ (a) and hmtASL^Met_CAU-Ψ₂₇ (b). NOSEY spectra of the hmtASL^Met_CAU in D₂O were recorded at 25° C with a mixing time of 400 ms. The H1′ and H5 to aromatic portion of the spectra are shown. The sequential H1′-aromatic connectivities are indicated, as well as the unusual NOEs connectivities between A₃₅H2 – A₃₇H1′ (blue) and A₃₅H8 – C₃₃H1′ (red).

Figure 4

Compilation of inter-nucleoside NOE connectivities. The inter-nucleoside NOEs observed in the loop residues 31 to 39 of the hmtASL^Met_CAU-Ψ₂₇;f⁵C₃₄ were both sequential and non-sequential. The sequential (i to i+1) inter-nucleosides NOEs are shown in blue and the nonsequential NOEs are shown in red. The hmtASL^Met_CAU-Ψ₂₇ exhibited comparable NOEs.

Figure 5

Structures of the hmtASL^Met_CAU-Ψ₂₇;f⁵C₃₄ and hmtASL^Met_CAU-Ψ₂₇. The ten lowest energy structures for the hmtASL^Met_CAU-Ψ₂₇;f⁵C₃₄ (a) and those for the hmtASL^Met_CAU-Ψ₂₇ (b) are superimposed. To illustrate differences in the degree to which the bases of a family of structures converge, the ribose and phosphodiester bond details are not shown.

Figure 6

Conformational differences and similarities in the anticodon loops of the f⁵C₃₄-modified and unmodified hmtASL^Met_CAU-Ψ₂₇. Stereo view of the average structures of the anticodon loops (residues C₃₂ to A₃₈) for (a) hmtASL^Met_CAU-Ψ₂₇ and (b) hmtASL^Met_CAU-Ψ₂₇;f⁵C₃₄. (c) The anticodon loops contain a C₃₂●C₃₈ intra-loop base pair in both the hmtASL^Met_CAU-Ψ₂₇;f⁵C₃₄ and the hmtASL^Met_CAU-Ψ₂₇. (d) Stereo view of the two average structures superimposed (green, hmtASL^Met-Ψ₂₇;f⁵C₃₄; and purple, the hmtASL^Met_CAU-Ψ₂₇).

Figure 7

AUG and AUA codon binding properties of the hmtASL^Met_CAU-Ψ₂₇;f⁵C₃₄ and hmtASL^Met_CAU-Ψ₂₇ in the P- and A-sites of the ribosome. (a) AUG and AUA codon binding of the hmtASL^Met_CAU-Ψ₂₇;f⁵C₃₄ and hmtASL^Met_CAU-Ψ₂₇ in the P-site of the ribosome. The E. coli 70S ribosome (5 pmoles), programmed with AUG in the P-site, was titrated with hmtASL^Met_CAU-Ψ₂₇;f⁵C₃₄ (▲) and with hmtASL^Met_CAU-Ψ₂₇ (■), or programmed with AUA and titrated with hmtASL^Met_CAU-Ψ₂₇;f⁵C₃₄ () and with hmtASL^Met_CAU-Ψ₂₇ (). The maximum hmtASL^Met_CUA bound was between 3-4 pmoles. (b) AUG and AUA codon binding properties of the hmtASL^Met_CAU-Ψ₂₇;f⁵C₃₄ and hmtASL^Met_CAU-Ψ₂₇ in the A-site of the ribosome. The E. coli 70S ribosome (5 pmoles), programmed with AUG in the A-site, was titrated with hmtASL^Met_CAU-Ψ₂₇;f⁵C₃₄ (▲) and with hmtASL^Met_CAU-Ψ₂₇ (■), or programmed with AUA and titrated with hmtASL^Met_CAU-Ψ₂₇;f⁵C₃₄ () and with hmtASL^Met_CAU-Ψ₂₇ (). The maximum hmtASL^Met_CUA bound was between 0.5-1.0 pmoles. In contrast to equilibrium binding studies, the time-dependent binding of the hmtASL^Met_CAU-Ψ₂₇;f⁵C₃₄ (▲) and hmtASL^Met_CAU-Ψ₂₇ (■) to AUG and AUA were conducted at the single concentration of ASL (0.5 μM), far too low to saturate the ribosomes. The hmtASL^Met_CAU-Ψ₂₇;f⁵C₃₄ and hmtASL^Met_CAU-Ψ₂₇ were bound to (c) AUG in the P-site of E. coli 70S ribosome, and to (d) AUA in the P-site). The hmtASL^Met_CAU-Ψ₂₇;f⁵C₃₄ and hmtASL^Met_CAU-Ψ₂₇ were bound to (e) AUG in the A-site of E. coli 70S ribosome, and to (f) AUA in the A-site.

Figure 8

Codon binding by hmtASL^Met_CAU, the native hmtRNA^Met_CAU and the unmodified transcript on the bovine mitochondrial ribosome. (a) AUG and AUA codon binding by the hmtASL^Met_CAU-Ψ₂₇;f⁵C₃₄ (f⁵C₃₄) and hmtASL^Met_CAU-Ψ₂₇ (unm) on the 55S bovine mitochondrial ribosome. The modification f⁵C₃₄ enhanced P-site binding of the AUG and AUA codons, and A-site binding to the AUG codon. However, A-site binding of the AUA codon by either ASL was not detectable. The binding (pmoles) of hmtASL^Met_CAU-Ψ₂₇;f⁵C₃₄ and hmtASL^Met_CAU-Ψ₂₇ are shown under conditions in which the ASLs are saturating. (b) The IF2-facilitated binding of bovine mtRNA^Met_CAU and its transcript to AUG and AUA in the P-site of the bovine mitochondrial ribosome. The bovine mitochondrial ribosome programmed with AUG was titrated with either mtRNA^Met_CAU (○) or its transcript (). Also, the AUA-programmed ribosome was titrated with either mtRNA^Met_CAU (◊) or its transcript ().

Figure 9

The f⁵C₃₄-modified hmtASL^Met_CAU-Ψ₂₇ binds the near-cognate codons AUU and AUC. The modification f⁵C₃₄ enabled hmtASL^Met_CAU-Ψ₂₇;f⁵C₃₄ to bind the near-cognate codons AUU and AUC, as well as AUA, in the P-site of the E. coli ribosome. The relative binding of non-cognate codons by hmtASL^Met_CAU-Ψ₂₇;f⁵C₃₄ (f⁵C₃₄) and hmtASL^Met_CAU-Ψ₂₇ (unm) are shown under conditions in which the ribosomes are saturated with ASLs, and the results internally normalized (percent). The binding of the valine codon GUA by hmtASL^Met_CAU-Ψ₂₇;f⁵C₃₄ (f⁵C₃₄) is an indication of the degree of non-specific binding in the assay.

Figure 10

Molecular dynamics simulation of the f⁵C₃₄●A base pair. The f⁵C₃₄●A base pair must occur on the mitochondrial ribosome when hmtRNA^Met_CAU reads the AUA codon in either the A-or P-sites. The base pairing of the 5′-monophosphate of f⁵C₃₄ with that of A under aqueous and neutral conditions was simulated with molecular dynamics, and the lowest energy structure is presented here. The resulting model indicated a possible bridging H₂O molecule that stabilizes the interaction.