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. 2023 Jul 11;18(10):2233–2239. doi: 10.1021/acschembio.3c00237

Practical Synthesis of N-Formylmethionylated Peptidyl-tRNA Mimics

Julia Thaler †, Egor A Syroegin ‡, Kathrin Breuker †, Yury S Polikanov ‡,§,∥,*, Ronald Micura †,∥,*
PMCID: PMC10594587  PMID: 37433044

Abstract

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Hydrolysis-resistant RNA-peptide conjugates that mimic peptidyl-tRNAs are frequently needed for structural and functional studies of protein synthesis in the ribosome. Such conjugates are accessible by chemical solid-phase synthesis, allowing for the utmost flexibility of both the peptide and the RNA sequence. Commonly used protection group strategies, however, have severe limitations with respect to generating the characteristic Nα-formylmethionyl terminus because the formyl group of the conjugate synthesized at the solid support is easily cleaved during the final basic deprotection/release step. In this study, we demonstrate a simple solution to the problem by coupling appropriately activated Nα-formyl methionine to the fully deprotected conjugate. The structural integrity of the obtained Nα-formylmethionyl conjugate—and hence the chemoselectivity of the reaction—were verified by Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry sequence analysis. Additionally, we confirmed the applicability of our procedure for structural studies by obtaining two structures of the ribosome in complex with either fMAI-nh-ACCA or fMFI-nh-ACCA in the P site and ACC-PMN in the A site of the bacterial ribosome at 2.65 and 2.60 Å resolution, respectively. In summary, our approach for hydrolysis-resistant Nα-formylated RNA-peptide conjugates is synthetically straightforward and opens up new avenues to explore ribosomal translation with high-precision substrate mimics.

Introduction

Protein biosynthesis is a vital cellular process accomplished by molecular machines known as ribosomes.1−3 During this process, also known as translation, tRNAs sequentially decode the mRNA harbored in the decoding center of the small ribosomal subunit, whereas their amino acid cargo assembles into a peptide chain in the peptidyl transferase center (PTC). The growing peptide chain exits the ribosome through a long narrow tunnel, which—for a long time—was assumed to only play a passive role in protein synthesis. In recent years, however, evidence arose that the tunnel can take an active role in folding of the nascent peptide chain4 and in the regulation of protein synthesis.5 For example, macrolide antibiotics bind in the tunnel of the bacterial ribosome and block certain growing peptides, consequently stalling and/or stopping translation.6−8 Moreover, many small organic compounds are known to modulate the rate of translation by interacting with the tunnel.5,9 Usually, these effects are associated with the nascent peptide of a particular sequence. A few such nascent-chain-arrested ribosome complexes with mRNA, tRNAs, nascent peptide chain, and the respective antibiotic have been structurally characterized. These studies shed the first light on the specific conformations and contacts that are responsible for the effects of the tunnel on translation.10−15 Related to this, the ribosomal synthesis of particular peptide sequences is intrinsically difficult, with successive prolines being the most prominent example; the restricted conformational freedom of polyprolines leads to steric interferences, which are considered to be partly responsible for these phenomena.16

In previous structural and biochemical studies, the peptidyl-tRNAs of stalled ribosomal complexes were commonly generated in cis by exploiting the natural peptidyl transferase activity of the ribosome and its ability to stall in the presence of a drug or a small molecule in the tunnel.10−13 This approach generates ribosome nascent chain complexes (RNCs) carrying peptidyl-tRNAs in the P site that contain native (wild-type) chains. What is an advantage on the one hand can be a drawback on the other because these biochemical preparations do not allow for variations in the peptide sequence. Such variations, however, are needed for reasons of comparison, e.g., to the nascent chain in the absence of the antibiotics that triggers stalling or as a consequence of a point mutation in the conserved stalling peptide sequences. Only recently, an important step toward such comparative structure determinations has been reported by capturing nonarrested nascent peptides in the prepeptidyl transfer state using hydrolysis-resistant peptidyl-tRNAs. This became possible by developing a procedure for preparing stable peptidyl-tRNA mimics, which carry an amide instead of naturally occurring ester linkages to prevent spontaneous deacylation of peptidyl-tRNAs during the time course of the experiments. Importantly, such nonhydrolyzable peptidyl-tRNAs are structurally indistinguishable from native tRNA substrates17 and are also active in transpeptidation when placed in the A site and combined with native aminoacyl-tRNA in the P site.18,19 Moreover, it has been shown that synthetic peptidyl-tRNAs and their short mimics can be efficiently complexed to the ribosome in vitro and yet represent a functionally significant state of the PTC.20,21 Therefore, amide-linked peptidyl-tRNA mimics represent a reasonable approximation of the reactive state, providing a reliable foundation for the mechanistic hypotheses.

The most convenient large-scale preparation of amide-linked full-length peptidyl-tRNAs follows a recent biochemical protocol20 of tRNAMet-tailing to replace the 3′-terminal regular adenosine-3′-OH of the CCA end with its amino-substituted adenosine-3′-NH2 analogue.22,23 Then, the tailed 3′-NH2-tRNAMet is enzymatically charged with cysteine by the aminoacyl-tRNAMet-synthetase,22,23 and finally, native chemical ligation of the thiobenzyl-activated N-formyl-methionyl peptide with cysteinyl-tRNA yields the desired product. The only drawback of this protocol is the restriction of the sequence to cysteine at the C-terminus of the peptide; prolyl-tRNA conjugates are, for instance, not accessible by this approach.

We, therefore, set out to advance a previously developed chemical solid-phase approach that produces complete 3′-amide-linked peptidyl-tRNA fragments 3 (Figure 1, left)24,25 that can subsequently be tailored into full-length tRNA with natural tRNA modifications.26,27 So far, the limitation of this approach has been the integrity of the characteristic Nα-formyl-methionine terminus because the formyl group of the conjugate assembled on the solid support 1 is typically cleaved during the basic deprotection/release step besides becoming oxidized (S to S=O) during RNA solid phase synthesis when P(III) is transformed into P(V) after each nucleoside coupling (Figure 1, left bottom, steps 2 to 3). Here, we present a simple solution to the problem by attaching an appropriately activated Nα-formyl methionine to fully deprotected conjugate 4 (Figure 1, right panel, steps 4 to 5). This enables efficient synthetic access of previously difficult-to-obtain peptidyl-tRNA mimics which carry the precise N-terminus of a nascent peptide chain, namely, N-formyl methionine.

Figure 1.

Figure 1

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Solid-phase synthesis (SPS) of peptidyl-tRNA mimics with an amide linkage based on support 1. Previous work has the limitation of lacking the characteristic Nα-formyl group at the N-terminal methionine (conjugate 3) because of deprotection under basic conditions (left panel). This work advances the earlier reported strategy by selective coupling of Nα-formyl methionine to fully deprotected conjugate 4 in solution to obtain target conjugate 5.

Results and Discussion

Chemical Synthesis of fMet-peptidyl-tRNA Mimics

The synthesis of N-formylmethionyl peptidyl-tRNA mimics starts from the previously developed solid support 1 (Figure 1, left panel). This support comprises the adenosine corresponding to the 3′ end A76 of a peptidyl-tRNA and the amide-linked amino acid corresponding to the C terminus of the peptidyl moiety of the same peptidyl-tRNA. The strength of the support is such that any amino acid (natural or modified) can be incorporated. Solid-phase peptide synthesis follows standard Nα-9-fluorenylmethoxycarbonyl (Fmoc) chemistry. Specifically, the side chains of Fmoc amino acid building blocks must be protected either by allyl groups (e.g., allyloxycarbony (alloc) for lysine) or by silyl-labile protecting groups (e.g., tert-butyldimethylsilyloxy (TBS) for serine) to be compatible with the protection strategy of RNA solid-phase synthesis that requires acidic conditions for the cleavage of the DMT group in each phosphoramidite coupling cycle. Consequently, typical tert-butyl protection or trityl protection of Fmoc amino acid side chains cannot be used. We also note that we preferably apply 2′-O-TBS instead of 2′-O-[(triisopropylsilyl) oxy]methyl (TOM) nucleoside building blocks to avoid any contamination with adducts arising from the reaction with the deprotection byproduct formaldehyde to the primary amino groups of the conjugate that might be encountered if high concentrations of conjugates (3 or 4) were used during treatment with TBAF in the final deprotection step.

To obtain target conjugate 5 harboring N-formylmethionyl termini, we first considered reagents that had been described in the literature for the formylation of the N-terminus of peptides. For instance, ethyl formate, p-nitrophenyl formate, and N-formylimidazole can be used for this purpose. These reagents are applied against the free NαH2 group (N-terminus), while the side-chain-protected peptide is still attached to the solid support. Then, during amino acid side chain deprotection and release of the peptide from the solid support, which occurs under acidic conditions, the Nα-formyl group remains stable. In contrast, synthetic peptidyl-RNA conjugates are deprotected under basic aqueous conditions and full (or partial) loss of the Nα-formyl group occurs; therefore, we did not further pursue this strategy.

More interesting is the recently introduced reagent of formyloxyacetoxyphenylmethane that has been successfully used for Nα-formylation of free peptides.28 However, we anticipated that, in the case of peptide-RNA conjugates, the nucleobase amino groups would also react in such a scenario. Therefore, we opted for an orthogonal strategy that avoids basic conditions and concomitant cleavage of the Nα-formyl group per se.

The starting point for our undertaking was readily accessible 3′-amide linked peptidyl-RNA conjugates of the general structure 4 (Figure 1).24,29−31 We wondered if fMet pentafluorophenyl (Pfp) acid ester is of sufficient reactivity and selectivity to form a peptide bond with the amino group of the N terminus (Figure 2A). Earlier work from our lab already demonstrated that fMet-OPfp can be used to couple fMet directly onto 3′-amino-3′-deoxy modified RNA.32 Here, incubation of conjugate 6 with the active ester indeed resulted in a nearly quantitative transformation to a slower migrating species (7), as reflected in the anion exchange HPLC traces (Figure 2B and C, Table 1). Isolation of this product and mass spectrometry revealed the expected molecular weight (Figure 2C). Importantly, we had no evidence for side products that could have emerged by reaction with the amino groups of the nucleobases A, C, and G or the 2′-hydroxyl functionalities of the oligoribonucleotide moiety. We therefore continued and prepared the diverse conjugates listed in Table 1 (see also Supporting Figure 1), following the pathway summarized in Figure 1 (right panel) to obtain mimics of the general chemical structure 5. The peptide sequences of the conjugates were chosen based on our earlier structural investigation of the context-specific action of the classic peptidyl transferase inhibitor chloramphenicol (12, 13),21 and, on the other hand, with the goal to contribute to the structural and mechanistic understanding of the protein synthesis with proline residues, which usually impede or even stall translation (9–11).33,34

Figure 2.

Figure 2

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Exemplary N-formylmethionylation of a peptidyl-RNA conjugate using N-fMet pentafluorophenyl ester. (A) Reaction scheme. (B) Anion exchange HPLC trace and ESI mass spectrum of conjugate 6 (starting material). (C) Anion exchange HPLC trace and ESI mass spectrum of fMet-conjugate (7) after conversion using the following conditions: concentration (of 6) = 100 μM, 20 mM fMet-OPfp, 100 mM Tris·HCl (pH 8) and DMSO (1:1), 37 °C, 15 min, 86%.

Table 1. Selection of Synthesized Conjugates.

# sequencea peptide (N to C terminus)-NH-3′-RNA-5′ molecular weight calcd [amu] molecular weightbfound [amu] yieldc [nmol; %]
7 fMSEAL-nh-ACCA 1765.5 1765.2 12 (87)
8 fMSEAdL-nh-ACCAd 1765.5 1765.0 12 (86)
9 fMPP-nh-ACCA 1559.3 1558.5 10 (91)
10 fMAPP-nh-ACCA 1630.4 1629.6 11 (97)
11 fMAAPP-nh-ACCA 1701.4 1700.6 13 (99)
12 fMAI-nh-ACCA 1549.3 1548.6 13 (97)
13 fMFI-nh-ACCA 1625.4 1624.5 9 (95)
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a

For the chemical structures, see Figure 1.

b

Determined by ESI mass spectrometry.

c

Total amount after purification; percentage (%) of conversion according to HPLC analysis is provided in parentheses.

d

Abbreviation dL specifies D configuration of leucine.

Top-Down MS Analysis of fMet-peptidyl-tRNA Mimics

To verify the sequence integrity of the fMet-peptidyl-RNA conjugates, we used electrospray ionization (ESI) Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry, which can determine the mass-to-charge ratio (m/z) of ions with very high precision by measuring the frequency of their cyclotron motion in a static magnetic field. A major strength of this approach is the direct sequencing of biopolymers through backbone cleavage by collisionally activated dissociation (CAD), which produces complementary b and y fragments for peptides and proteins and complementary c and y fragments for RNA (Figure 3A).35,36 This is typically achieved in the positive ion mode for peptides, while the negative ion mode is used for RNA. Thus, the challenge for top-down sequencing of RNA-peptide conjugates (as provided here) was to find conditions that allow the dissociation of the peptide and RNA backbone in the same experiment. We found that CAD of [M + 2H]2+ ions of conjugate 11 provided full sequence coverage in both peptide and RNA moieties (Figure 3B), in agreement with studies of DNA-peptide conjugates,37 and the fragment mass values unambiguously confirmed the sequence of fMAAPP-nh-ACCA.

Figure 3.

Figure 3

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Exemplary ESI FT-ICR mass spectrometric sequence analysis. (A) Collisionally activated dissociation (CAD) of (M + nH)n+ ions of peptides in the collision cell produces b and y fragment ions from amide backbone bond cleavage, and CAD of both (M – nH)n− and (M + nH)n+ ions of RNA produces c and y fragment ions from phosphodiester backbone bond cleavage. (B) Fragment-ion map illustrating sequence coverage from CAD of the peptidyl-RNA conjugate fMAAPP-nh-ACCA 11. CAD spectrum of the peptidyl-RNA conjugate with the most intensive signal assigned to undissociated (M + 2H)2+ ions. The insets show the isotopically resolved fragment signals with assignments of b and y fragments (cleavage in the peptide moiety) in red and assignments of c and y fragments (cleavage in the RNA moiety) in blue; calculated isotopic profiles for peptide and RNA fragments are indicated by red and blue open circles, respectively. The complete set of fragment MS signals from CAD of (M + 2H)2+ unambiguously confirm the conjugate sequence of fMAAPP-nh-ACCA.

fMet-peptidyl-tRNA Mimics Bound to Ribosomal PTC

To evaluate if formylation of the N-terminal methionine residue of the peptidyl-tRNA affects the overall conformation of the peptide in the PTC of the bacterial ribosome, we obtained two structures of the 70S ribosome in complex with ACC-Puromycin (PMN-CCA) and formylated fMAI-nh-ACCA (12) or fMFI-nh-ACCA (13) conjugates as the A- and P-site substrates, respectively (Figure 4). The observed electron density maps for both A- and P-site conjugates in both structures allowed unambiguous modeling of the short aminoacyl- and peptidyl-tRNA analogs (Figure 5). Moreover, the 2.65 Å (for fMAI-nh-ACCA analogue) and 2.60 Å (for fMFI-nh-ACCA analogue) spatial resolution of the obtained maps allowed us to directly visualize formyl groups at the N-termini of the ribosome-bound peptidyl-tRNA mimics (Figure 5A,C). Superpositioning of the new structures containing formylated versions of the peptidyl-tRNA mimics with those without formyl groups obtained before21 revealed no significant differences in the overall positions of the peptides (Figure 6).

Figure 4.

Figure 4

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Structures of formylated short tripeptidyl-tRNA analogs in the P site of the 70S ribosome. (A) Overview of the T. thermophilus 70S ribosome structures featuring short tRNA analogs viewed as a cross-cut section through the nascent peptide exit tunnel. The 30S subunit is shown in light yellow; the 50S subunit is in light blue. Ribosome-bound protein Y is colored in magenta. (B, C) 2Fo – Fc electron difference Fourier maps of PMN-CCA (green) and either formyl-MAI-tripeptidyl-tRNA (B, blue) or formyl-MFI-tripeptidyl-tRNA (C, teal) analogs. The refined models of short tRNA analogs are displayed in their respective electron density maps after the refinement (blue mesh). The overall resolution of the corresponding structures and the contour levels of the depicted electron density maps are shown at the bottom. (D) Superpositioning of the current 70S ribosome structures in complex with short tripeptidyl-tRNA analogs carrying fMAI (blue) and fMFI (teal) tripeptide sequences with each other. Note that the path of the growing polypeptide chain in the exit tunnel is not affected by the nature of the amino acid in the −1 position.

Figure 5.

Figure 5

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Electron density maps of the ribosome-bound formylated short peptidyl-tRNA analogs. (A, C) Unbiased Fo – Fc (gray and green mesh) and (B, D) 2Fo – Fc (blue mesh) electron difference Fourier maps of peptide moieties in the T. thermophilus 70S ribosome contoured at 2.5σ and 1.0σ, respectively. Gray mesh shows the Fo – Fc map after refinement with the entire peptidyl-tRNA analog omitted. Green mesh, reflecting the presence of formyl groups, shows the Fo – Fc electron density map after refinement with the nonformylated conjugates. The refined models of fMAI-nh-ACCA (A, B) or fMFI-nh-ACCA (C, D) conjugates are displayed in the corresponding electron density maps.

Figure 6.

Figure 6

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Comparison of the structures of formylated ribosome-bound peptidyl-tRNA analogs fMAI-nh-ACCA (A, blue) or fMFI-nh-ACCA (B, teal) in the P site with the previously reported structures of the same nonformylated conjugates. All structures were aligned based on domain V of the 23S rRNA.

Our previous studies showed that, in structural terms, these short tripeptidyl-tRNA analogs are indistinguishable from the natural full-length peptidyl-tRNAs in the P site of the ribosome.20,21 In particular, the orientation of the attacking α-amino group of the PMN-CCA (aminoacyl-tRNA mimic) relative to the carbonyl carbon of the P-site substrate are identical between the structures harboring full-length tRNAs versus those with short tRNA analogs.21 Thus, similar to our previous structures, both new structures represent the prepeptide bond formation state of the PTC (Figure 4B,C), in which peptide moieties of the peptidyl-tRNA analogs adopt similar beta-strand-like conformations, stabilized by the intricate network of H-bonds between their main-chain atoms and the universally conserved nucleotides U2506, G2061, and A2062 of the 23S rRNA.20,21 Taken together, our data suggest that the presence of the N-formyl group on tripeptidyl-tRNA mimics does not affect the position of the peptide in the ribosomal exit tunnel.

Conclusions

We have developed a straightforward approach to produce hydrolysis-resistant RNA-peptide conjugates that are Nα-formylmethionylated. Such conjugates mimic naturally occurring peptidyl-tRNAs and are needed for structural and functional studies of ribosomal translation to provide mechanistic insight into protein synthesis. One limitation in generating such conjugates by standard chemical solid-phase synthesis (SPS) has been the incompatibility of SPS with the introduction of a formyl group at the N-terminus of the peptide moiety. In this work, we overcome this challenge by finding the appropriate activation of fMet (in the form of its Pfp ester) to cleanly couple onto a free (unprotected) peptidyl-RNA conjugate. This transformation is equivalent to the direct formylation of a peptidyl-RNA conjugate precursor that would already contain methionine at the N-terminus; however, it has the advantage of a higher molecular weight increase, which makes HPLC product analysis straightforward because of more significant differences in retention times. Even more advantageous is that the presented approach masters the problem of methionine oxidation (thioether to sulfoxide; see, e.g., Figure 3 in reference (24)) that usually occurs during SPS of such conjugates because of repeated exposure to I2 solutions required for PIII-to-PV oxidation. In the new approach, the N-terminal methionine is coupled after solid-phase synthesis and hence is not exposed to oxidative reaction conditions. The method is particularly valuable because the synthetic precursor conjugates are broadly accessible, with hardly any sequence restrictions. The only limitation concerns lysines that contain a primary amino group at their side chains and that are formylmethionylated without a proper protection strategy. Conceptually, lysine-containing conjugates are accessible by this approach only if a photolabile protection group is applied at the N(ε) position that becomes finally cleaved after fMet has been coupled to the N peptide terminus.

In summary, our path toward hydrolysis-resistant Nα-formylated RNA-peptide conjugates opens up new avenues to explore protein synthesis with high-precision substrate mimics. Although the present showcase of fMFI and fMAI conjugates indicates that the formylation does not alter the binding mode and conformation of these particular peptides, and thus, the biological impact of these findings is relatively modest, it is important that this has been assessed directly, and also, this may not be the case for other peptides.

Methods

Preparation of Peptidyl-RNA Precursors 4 with NH2 Termini

All peptidyl-RNA conjugates of type 4 were produced and purified following references (24), (29), and (30). The assembly of the conjugates was based on Fmoc peptide solid-phase synthesis and RNA solid-phase synthesis using 2′-O-[(triisopropylsilyl) oxy]methyl (TOM)- or 2′-O-[tert-butyldimetylsilyl (TBDMS)-protected nucleoside building blocks.

Additional Remark on the Deprotection of Peptidyl-RNAs Containing Allyl Protected Glutamic Acid. Allyl deprotection was performed in analogy to reference (29). After conjugate assembly, the solid support was treated in the synthesis cartridge with a solution of N-methyl morpholine (37 μL, 0.34 mmol) and acetic acid (37 μL, 65 mmol) in chloroform (amylene stabilized; 1 mL). After the addition of tetrakis(triphenylphosphine) palladium(0) (12 mg, 0.01 mmol), the suspension was agitated for 5 h at RT. Subsequently, the solid support was washed with chloroform (3 × 2 mL), dried under a vacuum, and subjected to standard acyl and 2′-O-silyl deprotection; see references (24), (29), and (30).

Synthesis of fMet Peptidyl-tRNAs 5 (i.e., Conjugates 7 to 13)

N-Formyl-l-methionine pentafluorophenylester was synthesized as described in references (32). One equivalent of 3′-amino-3′-deoxyoligoribonucleotides 4 (final concentration = 0.1 mM) and 200 equiv of N-formyl-l-methionine pentafluorophenylester (final concentration = 20 mM) were dissolved in 100 mM Tris–HCl (pH 8.0) and dimethyl sulfoxide (1/1, v/v). The typical total reaction volume amounted to 150 μL. After 15 min at 37 °C, the reaction mixture was diluted with 450 μL water and directly applied on a size-exclusion chromatography column (GE Healthcare, HiPrep 26/10 Desalting, 2.6 × 10 cm, Sephadex G25). By elution with H2O, the conjugate-containing fractions were collected and evaporated to dryness, and the residue was dissolved in H2O (1 mL). Analysis of the crude products was performed by anion-exchange chromatography on a Dionex DNAPac PA-100 column (4 × 250 mm) at 60 °C (flow rate: 1 mL min–1; eluent A, 25 mM Tris-HCl (pH 8.0) and 20 mM NaClO4 in 20% aqueous acetonitrile; eluent B, 25 mM Tris-HCl (pH 8.0) and 0.60 M NaClO4 in 20% aqueous acetonitrile; gradient: 0–35% B in A within 30 min; UV detection at λ = 260 nm).

Purification of fMet peptidyl-tRNAs 5

If the conversion reaction yielded less than 80% of the fMet peptidyl-RNA, the conjugates were additionally purified on a semipreparative Dionex DNAPac PA-100 column (9 × 250 mm) at 60 °C with a flow rate of 2 mL min–1 (for eluents, see the section Synthesis of fMet Peptidyl-tRNAs 5). Fractions containing the conjugate were concentrated to near dryness and diluted with 0.1 M (Et3NH)+HCO3– and loaded on a C18 SepPak Plus cartridge (Waters, Millipore), washed with H2O, and eluted with H2O/CH3CN (1:1). Conjugate-containing fractions were evaporated to dryness and dissolved in H2O (1 mL). The quality of the purified conjugate was analyzed by analytical anion-exchange chromatography (for conditions, see the section Synthesis of fMet Peptidyl-tRNAs 5). The molecular weight of the synthesized conjugate was confirmed by liquid chromatography-electron ionization (LC-ESI) mass spectrometry (Table 1). Yields were determined by UV photometrical analysis of conjugate solutions.

FT-ICR Mass Spectrometry Analysis of fMet Peptidyl-RNA Products

Experiments were performed on a 7T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer (Bruker APEX ultra) equipped with an ESI source and a collision cell through which a flow of Ar gas was maintained for CAD. Peptide-RNA conjugates were electrosprayed (flow rate 1.5 μL/min) from 1 μM solutions in 1:1 H2O/CH3OH vol/vol with 1% acetic acid as an additive. Methanol (Acros) and acetic acid (Fisher Scientific) were HPLC grade, and H2O was purified to 18 MΩ·cm at RT using a Milli-Q system (Millipore). RNA concentration was determined by UV absorption at 260 nm using a NanoPhotometer (Implen). Prior to dissociation by CAD, the (M + nH)n+ ions under study were isolated in a linear quadrupole; for a more detailed description of the experimental setup for CAD, see reference (38).

Crystallographic Structure Determination

X-ray crystal structures of 70S ribosomes from Thermus thermophilus in complex with protein Y and short tRNA mimics were determined as described previously, see reference (21). The statistics of data collection and refinement are compiled in Supporting Information Table 1. All figures showing atomic models were rendered using PyMol software (www.pymol.org).

Acknowledgments

We thank C. Kreutz (University of Innsbruck) for NMR spectroscopic support and D. Fellner (University of Innsbruck) and U. Schober (University of Innsbruck) for technical support. We thank the staff at NE-CAT beamlines 24ID-C and 24ID-E for help with X-ray diffraction data collection, especially M. Capel, F. Murphy, S. Banerjee, I. Kourinov, D. Neau, J. Schuermann, N. Sukumar, A. Lynch, J. Withrow, K. Perry, A. Kaya, and C. Salbego. This work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health [P30-GM124165 to NE-CAT]. The Eiger 16M detector on the 24-ID-E beamline is funded by an NIH-ORIP HEI grant [S10-OD021527 to NE-CAT]. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. This work was supported by the Austrian Science Fund FWF [P31691, F8011-B to R.M.; P30087, P36011 to K.B.], the Austrian Research Promotion Agency FFG [West Austrian Bio NMR 858017 to R.M.], the National Institute of General Medical Sciences of the National Institutes of Health [R01-GM132302 to Y.S.P.], the National Institute of Allergy and Infectious Diseases of the National Institutes of Health [R01-AI162961 and R21-AI163466 to Y.S.P.], and the Illinois State startup funds [to Y.S.P.]. The funders had no role in study design, data collection and analysis, decision to publish, or manuscript preparation.

Data Availability Statement

Coordinates and structure factors were deposited in the RCSB Protein Data Bank with accession codes 8T8B for the T. thermophilus 70S ribosome in complex with protein Y, A-site aminoacyl-tRNA analog ACC-Pmn, and P-site peptidyl-tRNA analog fMAI-nh-ACCA; 8T8C for the T. thermophilus 70S ribosome in complex with protein Y, A-site aminoacyl-tRNA analog ACC-Pmn, and P-site peptidyl-tRNA analog fMAI-nh-ACCA;

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschembio.3c00237.

  • Statistics of crystallographic data collection/refinement and further characterization data of conjugates (PDF)

Author Contributions

⊥ These authors contributed equally. The manuscript was written through the contributions of all authors. All authors have approved the final version of the manuscript.

Open Access is funded by the Austrian Science Fund (FWF).

The authors declare no competing financial interest.

Special Issue

Published as part of ACS Chemical Biologyvirtual special issue “Nucleic Acid Regulation”.

Supplementary Material

cb3c00237_si_001.pdf (340.2KB, pdf)

References

  1. Wilson D. N.; Nierhaus K. H. The weird and wonderful world of bacterial ribosome regulation. Crit. Rev. Biochem. Mol. Biol. 2007, 42, 187–219. 10.1080/10409230701360843. [DOI] [PubMed] [Google Scholar]
  2. Munro J. B.; Sanbonmatsu K. Y.; Spahn C. M.; Blanchard S. C. Navigating the ribosome’s metastable energy landscape. Trends Biochem. Sci. 2009, 34, 390–400. 10.1016/j.tibs.2009.04.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Trappl K.; Polacek N. The ribosome: a molecular machine powered by RNA. Met. Ions Life Sci. 2011, 9, 253–275. 10.1039/9781849732512-00253. [DOI] [PubMed] [Google Scholar]
  4. Bhushan S.; Gartmann M.; Halic M.; Armache J. P.; Jarasch A.; Mielke T.; Berninghausen O.; Wilson D. N.; Beckmann R. α-Helical nascent polypeptide chains visualized within distinct regions of the ribosomal exit tunnel. Nat. Struct. Mol. Biol. 2010, 17, 313–317. 10.1038/nsmb.1756. [DOI] [PubMed] [Google Scholar]
  5. Ito K.; Chiba S. Arrest peptides: cis-acting modulators of translation. Annu. Rev. Biochem. 2013, 82, 171–202. 10.1146/annurev-biochem-080211-105026. [DOI] [PubMed] [Google Scholar]
  6. Vazquez-Laslop N.; Mankin A. S. How macrolide antibiotics work. Trends Biochem. Sci. 2018, 43, 668–684. 10.1016/j.tibs.2018.06.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Wilson D. N. Ribosome-targeting antibiotics and mechanisms of bacterial resistance. Nat. Rev. Microbiol. 2014, 12, 35–48. 10.1038/nrmicro3155. [DOI] [PubMed] [Google Scholar]
  8. Lin J.; Zhou D.; Steitz T. A.; Polikanov Y. S.; Gagnon M. G. Ribosome-targeting antibiotics: modes of action, mechanisms of resistance, and implications for drug design. Annu. Rev. Biochem. 2018, 87, 451–478. 10.1146/annurev-biochem-062917-011942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Seip B.; Innis C. A. How widespread is metabolite sensing by ribosome-arresting nascent peptides?. J. Mol. Biol. 2016, 428, 2217–2227. 10.1016/j.jmb.2016.04.019. [DOI] [PubMed] [Google Scholar]
  10. Arenz S.; Bock L. V.; Graf M.; Innis C. A.; Beckmann R.; Grubmüller H.; Vaiana A. C.; Wilson D. N. A combined cryo-EM and molecular dynamics approach reveals the mechanism of ErmBL-mediated translation arrest. Nat. Commun. 2016, 7, 12026. 10.1038/ncomms12026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Arenz S.; Meydan S.; Starosta A. L.; Berninghausen O.; Beckmann R.; Vázquez-Laslop N.; Wilson D. N. Drug sensing by the ribosome induces translational arrest via active site perturbation. Mol. Cell 2014, 56, 446–452. 10.1016/j.molcel.2014.09.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Arenz S.; Ramu H.; Gupta P.; Berninghausen O.; Beckmann R.; Vázquez-Laslop N.; Mankin A. S.; Wilson D. N. Molecular basis for erythromycin-dependent ribosome stalling during translation of the ErmBL leader peptide. Nat. Commun. 2014, 5, 3501. 10.1038/ncomms4501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Beckert B.; Leroy E. C.; Sothiselvam S.; Bock L. V.; Svetlov M. S.; Graf M.; Arenz S.; Abdelshahid M.; Seip B.; Grubmüller H.; Mankin A. S.; Innis C. A.; Vázquez-Laslop N.; Wilson D. N. Structural and mechanistic basis for translation inhibition by macrolide and ketolide antibiotics. Nat. Commun. 2021, 12, 4466. 10.1038/s41467-021-24674-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Herrero Del Valle A.; Seip B.; Cervera-Marzal I.; Sacheau G.; Seefeldt A. C.; Innis C. A. Ornithine capture by a translating ribosome controls bacterial polyamine synthesis. Nat. Microbiol. 2020, 5, 554–561. 10.1038/s41564-020-0669-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Su T.; Cheng J.; Sohmen D.; Hedman R.; Berninghausen O.; von Heijne G.; Wilson D. N.; Beckmann R. The force-sensing peptide VemP employs extreme compaction and secondary structure formation to induce ribosomal stalling. eLife 2017, 6, e25642 10.7554/eLife.25642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Doerfel L. K.; Wohlgemuth I.; Kothe C.; Peske F.; Urlaub H.; Rodnina M. V. EF-P is essential for rapid synthesis of proteins containing consecutive proline residues. Science 2013, 339, 85–88. 10.1126/science.1229017. [DOI] [PubMed] [Google Scholar]
  17. Loveland A. B.; Demo G.; Korostelev A. A. Cryo-EM of elongating ribosome with EF-Tu*GTP elucidates tRNA proofreading. Nature 2020, 584, 640–645. 10.1038/s41586-020-2447-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Polikanov Y. S.; Steitz T. A.; Innis C. A. A proton wire to couple aminoacyl-tRNA accommodation and peptide-bond formation on the ribosome. Nat. Struct. Mol. Biol. 2014, 21, 787–793. 10.1038/nsmb.2871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fraser T. H.; Rich A. Synthesis and aminoacylation of 3′-amino- 3′-deoxy transfer RNA and its activity in ribosomal protein synthesis. Proc. Natl. Acad. Sci. USA 1973, 70, 2671–2675. 10.1073/pnas.70.9.2671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Syroegin E. A.; Aleksandrova E. V.; Polikanov Y. S. Insights into the ribosome function from the structures of non-arrested ribosome-nascent chain complexes. Nat. Chem. 2023, 15, 143–153. 10.1038/s41557-022-01073-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Syroegin E. A.; Flemmich L.; Klepacki D.; Vazquez-Laslop N.; Micura R.; Polikanov Y. S. Structural basis for the context-specific action of the classic peptidyl transferase inhibitor chloramphenicol. Nat. Struct. Mol. Biol. 2022, 29, 152–161. 10.1038/s41594-022-00720-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gamper H.; Hou Y. M. tRNA 3′-amino-tailing for stable amino acid attachment. RNA 2018, 24, 1878–1885. 10.1261/rna.068015.118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Svetlov M. S.; Syroegin E. A.; Aleksandrova E. V.; Atkinson G. C.; Gregory S. T.; Mankin A. S.; Polikanov Y. S. Structure of Erm-modified 70S ribosome reveals the mechanism of macrolide resistance. Nat. Chem. Biol. 2021, 17, 412–420. 10.1038/s41589-020-00715-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Moroder H.; Steger J.; Graber D.; Fauster K.; Trappl K.; Marquez V.; Polacek N.; Wilson D. N.; Micura R. Non-hydrolyzable RNA-peptide conjugates: a powerful advance in the synthesis of mimics for 3′-peptidyl tRNA termini. Angew. Chem., Int. Ed. 2009, 48, 4056–4060. 10.1002/anie.200900939. [DOI] [PubMed] [Google Scholar]
  25. Terenzi S.; Biała E.; Nguyen-Trung N. Q.; Strazewski P. Amphiphilic 3′-peptidyl-RNA conjugates. Angew. Chem., Int. Ed. 2003, 42, 2909–2912. 10.1002/anie.200350926. [DOI] [PubMed] [Google Scholar]
  26. Graber D.; et al. Reliable semi-synthesis of hydrolysis-resistant 3′-peptidyl-tRNA conjugates containing genuine tRNA modifications. Nucleic Acids Res. 2010, 38, 6796–6802. 10.1093/nar/gkq508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Graber D.; Trappl K.; Steger J.; Geiermann A. S.; Rigger L.; Moroder H.; Polacek N.; Micura R. Deoxyribozyme-based, semisynthetic access to stable peptidyl-tRNAs exemplified by tRNAVal carrying a macrolide antibiotic resistance peptide. Methods Mol. Biol. 2012, 848, 201–213. 10.1007/978-1-61779-545-9_13. [DOI] [PubMed] [Google Scholar]
  28. Chapman R. S. L.; Lawrence R.; Williams J. M. J.; Bull S. D. Formyloxyacetoxyphenylmethane as an N-Formylating Reagent for Amines, Amino Acids, and Peptides. Org. Lett. 2017, 19, 4908–4911. 10.1021/acs.orglett.7b02382. [DOI] [PubMed] [Google Scholar]
  29. Rigger L.; Schmidt R. L.; Holman K. M.; Simonović M.; Micura R. The synthesis of methylated, phosphorylated, and phosphonated 3′-aminoacyl-tRNA(Sec) mimics. Chem.—Eur. J. 2013, 19, 15872–15878. 10.1002/chem.201302188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Steger J.; Micura R. Functionalized polystyrene supports for solid-phase synthesis of glycyl-, alanyl-, and isoleucyl-RNA conjugates as hydrolysis-resistant mimics of peptidyl-tRNAs. Bioorg. Med. Chem. 2011, 19, 5167–5174. 10.1016/j.bmc.2011.07.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Krasheninina O. A.; Thaler J.; Erlacher M. D.; Micura R. Amine-to-Azide Conversion on Native RNA via Metal-Free Diazotransfer Opens New Avenues for RNA Manipulations. Angew. Chem., Int. Ed. 2021, 60, 6970–6974. 10.1002/anie.202015034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Steger J.; Graber D.; Moroder H.; Geiermann A. S.; Aigner M.; Micura R. Efficient access to nonhydrolyzable initiator tRNA based on the synthesis of 3′-azido-3′-deoxyadenosine RNA. Angew. Chem., Int. Ed. 2010, 49, 7470–7472. 10.1002/anie.201003424. [DOI] [PubMed] [Google Scholar]
  33. Melnikov S.; Mailliot J.; Rigger L.; Neuner S.; Shin B. S.; Yusupova G.; Dever T. E.; Micura R.; Yusupov M. Molecular insights into protein synthesis with proline residues. EMBO Rep. 2016, 17, 1776–1784. 10.15252/embr.201642943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Huter P.; Arenz S.; Bock L. V.; Graf M.; Frister J. O.; Heuer A.; Peil L.; Starosta A. L.; Wohlgemuth I.; Peske F.; Nováček J.; Berninghausen O.; Grubmüller H.; Tenson T.; Beckmann R.; Rodnina M. V.; Vaiana A. C.; Wilson D. N. Structural Basis for Polyproline-Mediated Ribosome Stalling and Rescue by the Translation Elongation Factor EF-P. Mol. Cell 2017, 68, 515–527. 10.1016/j.molcel.2017.10.014. [DOI] [PubMed] [Google Scholar]
  35. Han X.; Jin M.; Breuker K.; McLafferty F. W. Extending top-down mass spectrometry to proteins with masses greater than 200 kDas. Science 2006, 314, 109–112. 10.1126/science.1128868. [DOI] [PubMed] [Google Scholar]
  36. Glasner H.; Riml C.; Micura R.; Breuker K. Label-free, direct localization and relative quantitation of the RNA nucleobase methylations m6A, m5C, m3U, and m5U by top-down mass spectrometry. Nucleic Acids Res. 2017, 45, 8014–8025. 10.1093/nar/gkx470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Jensen O. N.; Kulkarni S.; Aldrich J. V.; Barofsky D. F. Charac-terization of peptide-oligonucleotide heteroconjugates by mass spectrometry. Nucleic Acids Res. 1996, 24, 3866–3872. 10.1093/nar/24.19.3866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Taucher M.; Rieder U.; Breuker K. Minimizing base loss and internal fragmentation in collisionally activated dissociation of multiply deprotonated RNA. J. Am. Soc. Mass Spectrom. 2010, 21, 278–285. 10.1016/j.jasms.2009.10.010. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

cb3c00237_si_001.pdf (340.2KB, pdf)

Data Availability Statement

Coordinates and structure factors were deposited in the RCSB Protein Data Bank with accession codes 8T8B for the T. thermophilus 70S ribosome in complex with protein Y, A-site aminoacyl-tRNA analog ACC-Pmn, and P-site peptidyl-tRNA analog fMAI-nh-ACCA; 8T8C for the T. thermophilus 70S ribosome in complex with protein Y, A-site aminoacyl-tRNA analog ACC-Pmn, and P-site peptidyl-tRNA analog fMAI-nh-ACCA;

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