tRNA Substrates Research Articles

Translational roles of the C75 2\u2032OH in an in vitro tRNA transcript at the ribosomal A, P and E sites

Aminoacyl-tRNAs containing a deoxy substitution in the penultimate nucleotide (C75 2′OH → 2′H) have been widely used in translation for incorporation of unnatural amino acids (AAs). However, this supposedly innocuous modification surprisingly increased peptidyl-tRNAAlaugc drop off in biochemical assays of successive incorporations. Here we predict the function of this tRNA 2′OH in the ribosomal A, P and E sites using recent co-crystal structures of ribosomes and tRNA substrates and test these structure-function models by systematic kinetics analyses. Unexpectedly, the C75 2′H did not affect A- to P-site translocation nor peptidyl donor activity of tRNAAlaugc. Rather, the peptidyl acceptor activity of the A-site Ala-tRNAAlaugc and the translocation of the P-site deacylated tRNAAlaugc to the E site were impeded. Delivery by EF-Tu was not significantly affected. This broadens our view of the roles of 2′OH groups in tRNAs in translation.

Structural basis for substrate binding and catalytic mechanism of a human RNA:m5C methyltransferase NSun6.

5-methylcytosine (m5C) modifications of RNA are ubiquitous in nature and play important roles in many biological processes such as protein translational regulation, RNA processing and stress response. Aberrant expressions of RNA:m5C methyltransferases are closely associated with various human diseases including cancers. However, no structural information for RNA-bound RNA:m5C methyltransferase was available until now, hindering elucidation of the catalytic mechanism behind RNA:m5C methylation. Here, we have solved the structures of NSun6, a human tRNA:m5C methyltransferase, in the apo form and in complex with a full-length tRNA substrate. These structures show a non-canonical conformation of the bound tRNA, rendering the base moiety of the target cytosine accessible to the enzyme for methylation. Further biochemical assays reveal the critical, but distinct, roles of two conserved cysteine residues for the RNA:m5C methylation. Collectively, for the first time, we have solved the complex structure of a RNA:m5C methyltransferase and addressed the catalytic mechanism of the RNA:m5C methyltransferase family, which may allow for structure-based drug design toward RNA:m5C methyltransferase–related diseases.

Madumycin II inhibits peptide bond formation by forcing the peptidyl transferase center into an inactive state.

The emergence of multi-drug resistant bacteria is limiting the effectiveness of commonly used antibiotics, which spurs a renewed interest in revisiting older and poorly studied drugs. Streptogramins A is a class of protein synthesis inhibitors that target the peptidyl transferase center (PTC) on the large subunit of the ribosome. In this work, we have revealed the mode of action of the PTC inhibitor madumycin II, an alanine-containing streptogramin A antibiotic, in the context of a functional 70S ribosome containing tRNA substrates. Madumycin II inhibits the ribosome prior to the first cycle of peptide bond formation. It allows binding of the tRNAs to the ribosomal A and P sites, but prevents correct positioning of their CCA-ends into the PTC thus making peptide bond formation impossible. We also revealed a previously unseen drug-induced rearrangement of nucleotides U2506 and U2585 of the 23S rRNA resulting in the formation of the U2506•G2583 wobble pair that was attributed to a catalytically inactive state of the PTC. The structural and biochemical data reported here expand our knowledge on the fundamental mechanisms by which peptidyl transferase inhibitors modulate the catalytic activity of the ribosome.

A Functional Role for the Monomethylated Gln-51 and Lys-53 Residues of the 49GGQTK53 Motif of eL42 from Human 80S Ribosomes.

Background:We have previously demonstrated that the eukaryote-specific ribosomal protein eL42 of the human 80S ribosome contains seven monomethylated residues, among which are the Gln-51 and Lys-53 residues contained in the 47GFGGQTK53 sequence conserved in all eukaryotic 80S ribosomes. This sequence contains the methylated and universally conserved GGQ motif common for all class-1 translation termination factors responsible for stop codon recognition and for triggering the hydrolysis of the P site-bound peptidyl-tRNA. We have also recently reported a model of ribosomal ternary eL42-tRNA-eRF1 complex where specific regions of all three macromolecules (the comparably flexible GGQ domains of eRF1 and eL42 and the CCA-arm of tRNA) are involved in interactions.Method:Here, we have studied the interactions between recombinant eL42 and eRF1 proteins and the tRNA substrate by means of the Biacore assay, using the wild-type eL42 protein, the eL42-Δ(GGQTK) mutant (the eL42 protein whose GGQTK motif has been deleted), the single Q51E and K53Q mutants (eL42-Q51E and eL42-K53Q, respectively), as well as the double Q51A/K53A mutant (eL42-Q51A/K53A).Results:Our results show that the monomethylated Gln-51 and Lys-53 residues contained in the 47GFGGQTK53 sequence of eL42 and the monomethylated GGQ motif of eRF1 represents the sites of interaction between these two proteins through hydrophobic contacts between methyl groups. We also demonstrate that the interactions between eL42 and tRNA or 28S rRNA are characterized by strong binding affinities (KD values in the nanomolar or picomolar range, respectively) which argue for specific interactions. Strong interactions between eL42 and tRNA are likely to be responsible for the decrease in the poly(U)-dependent poly(Phe) synthesis activity of human 80S or E. coli 70S ribosomes in the presence of added human recombinant eL42. It is proposed that the decrease of the activity of the ribosome is caused by the sequestration of the substrate Phe-tRNAPhe by the added eL42 protein.Conclusion:Interactions between the monomethylated Gln-51 and Lys-53 residues of the 49GGQTK53 motif of the human eL42 protein and the methylated GGQ motif of eRF1 are likely to play a functional role on translating human 80S ribosomes.

Trm5 and TrmD: Two Enzymes from Distinct Origins Catalyze the Identical tRNA Modification, m¹G37.

The N1-atom of guanosine at position 37 in transfer RNA (tRNA) is methylated by tRNA methyltransferase 5 (Trm5) in eukaryotes and archaea, and by tRNA methyltransferase D (TrmD) in bacteria. The resultant modified nucleotide m1G37 positively regulates the aminoacylation of the tRNA, and simultaneously functions to prevent the +1 frameshift on the ribosome. Interestingly, Trm5 and TrmD have completely distinct origins, and therefore bear different tertiary folds. In this review, we describe the different strategies utilized by Trm5 and TrmD to recognize their substrate tRNAs, mainly based on their crystal structures complexed with substrate tRNAs.

Biochemical Characterization of AP Lyase and m6A Demethylase Activities of Human AlkB Homologue 1 (ALKBH1).

Alkbh1 is one of nine mammalian homologues of Escherichia coli AlkB, a 2-oxoglutarate-dependent dioxygenase that catalyzes direct DNA repair by removing alkyl lesions from DNA. Six distinct enzymatic activities have been reported for Alkbh1, including hydroxylation of variously methylated DNA, mRNA, tRNA, or histone substrates along with the cleavage of DNA at apurinic/apyrimidinic (AP) sites followed by covalent attachment to the 5'-product. The studies described here extend the biochemical characterization of two of these enzymatic activities using human ALKBH1: the AP lyase and 6-methyl adenine DNA demethylase activities. The steady-state and single-turnover kinetic parameters for ALKBH1 cleavage of AP sites in DNA were determined and shown to be comparable to those of other AP lyases. The α,β-unsaturated aldehyde of the 5'-product arising from DNA cleavage reacts predominantly with C129 of ALKBH1, but secondary sites also generate covalent adducts. The 6-methyl adenine demethylase activity was examined with a newly developed assay using a methylation-sensitive restriction endonuclease, and the enzymatic rate was found to be very low. Indeed, the demethylase activity was less than half that of the AP lyase activity when ALKBH1 samples were assayed using identical buffer conditions. The two enzymatic activities were examined using a series of site-directed variant proteins, revealing the presence of distinct but partially overlapping active sites for the two reactions. We postulate that the very low 6-methyl adenine oxygenase activity associated with ALKBH1 is unlikely to represent the major function of the enzyme in the cell, while the cellular role of the lyase activity (including its subsequent covalent attachment to DNA) remains uncertain.

Requirement of the RNA-binding protein SmpB during intracellular growth of Listeria monocytogenes

Bacterial trans-translation is the main quality control mechanism employed to relieve stalled ribosomes. Trans-translation is mediated by the small protein B (SmpB) and transfer-mRNA (tmRNA) ribonucleoprotein complex, which interacts with translational complexes stalled at the 3′ end of non-stop mRNAs to release the stalled ribosomes thereby targeting the nascent polypeptides and truncated mRNAs for degradation. The trans-translation system exists with a few exceptions in all bacteria.In the present study, we assessed the contribution of SmpB to the growth and virulence of Listeria monocytogenes, a human intracellular food-borne pathogen that colonizes host tissues to cause severe invasive infections. A smpB knockout significantly decreased the intracellular growth rate of L. monocytogenes during infection of murine macrophages. In addition, the mutant strain was attenuated for virulence when examined with the Galleria mellonella larvae killing assay and the organ colonisation model of mice following infection.Proteomic analysis of whole cell extracts of ΔsmpB deletion mutant revealed elevated protein levels of several proteins involved in ribosome assembly and interaction with tRNA substrates. These included the elongation factor Tu [EF-Tu] which promotes the GTP-dependent binding of aminoacyl-tRNA to the A-site of ribosomes during protein biosynthesis as well as the CysK which is known to interact with bacterial toxins that cleave tRNA substrates. The data presented here shed light on the role of SmpB and trans-translation during intracellular growth of L. monocytogenes.

S. cerevisiae Trm140 has two recognition modes for 3-methylcytidine modification of the anticodon loop of tRNA substrates.

The 3-methylcytidine (m3C) modification is ubiquitous in eukaryotic tRNA, widely found at C32 in the anticodon loop of tRNAThr, tRNASer, and some tRNAArg species, as well as in the variable loop (V-loop) of certain tRNASer species. In the yeast Saccharomyces cerevisiae, formation of m3C32 requires Trm140 for six tRNA substrates, including three tRNAThr species and three tRNASer species, whereas in Schizosaccharomyces pombe, two Trm140 homologs are used, one for tRNAThr and one for tRNASer. The occurrence of a single Trm140 homolog is conserved broadly among Ascomycota, whereas multiple Trm140-related homologs are found in metazoans and other fungi. We investigate here how S. cerevisiae Trm140 protein recognizes its six tRNA substrates. We show that Trm140 has two modes of tRNA substrate recognition. Trm140 recognizes G35-U36-t6A37 of the anticodon loop of tRNAThr substrates, and this sequence is an identity element because it can be used to direct m3C modification of tRNAPhe. However, Trm140 recognition of tRNASer substrates is different, since their anticodons do not share G35–U36 and do not have any nucleotides in common. Rather, specificity of Trm140 for tRNASer is achieved by seryl-tRNA synthetase and the distinctive tRNASer V-loop, as well as by t6A37 and i6A37. We provide evidence that all of these components are important in vivo and that seryl-tRNA synthetase greatly stimulates m3C modification of tRNASer(CGA) and tRNASer(UGA) in vitro. In addition, our results show that Trm140 binding is a significant driving force for tRNA modification and suggest separate contributions from each recognition element for the modification.

The RNA methyltransferase Dnmt2 methylates DNA in the structural context of a tRNA

ABSTRACTThe amino acid sequence of Dnmt2 is very similar to the catalytic domains of bacterial and eukaryotic DNA-(cytosine 5)-methyltransferases, but it efficiently catalyzes tRNA methylation, while its DNA methyltransferase activity is the subject of controversial reports with rates varying between zero and very weak. By using composite nucleic acid molecules as substrates, we surprisingly found that DNA fragments, when presented as covalent DNA-RNA hybrids in the structural context of a tRNA, can be more efficiently methylated than the corresponding natural tRNA substrate. Furthermore, by stepwise development of tRNAAsp, we showed that this natural Dnmt2 substrate could be engineered to employ RNAs that act like guide RNAs in vitro. The 5’-half of tRNAAsp was able to efficiently guide methylation toward a single stranded tRNA fragment as would result from tRNA cleavage by tRNA specific nucleases. In a more artificial setting, a composite system of guide RNAs could ultimately be engineered to enable the enzyme to perform cytidine methylation on single stranded DNA in vitro.

Sequence-specific and Shape-selective RNA Recognition by the Human RNA 5-Methylcytosine Methyltransferase NSun6

Human NSun6 is an RNA methyltransferase that catalyzes the transfer of the methyl group from S-adenosyl-l-methionine (SAM) to C72 of tRNAThr and tRNACys In the current study, we used mass spectrometry to demonstrate that human NSun6 indeed introduces 5-methylcytosine (m5C) into tRNA, as expected. To further reveal the tRNA recognition mechanism of human NSun6, we measured the methylation activity of human NSun6 and its kinetic parameters for different tRNA substrates and their mutants. We showed that human NSun6 requires a well folded, full-length tRNA as its substrate. In the acceptor region, the CCA terminus, the target site C72, the discriminator base U73, and the second and third base pairs (2:71 and 3:70) of the acceptor stem are all important RNA recognition elements for human NSun6. In addition, two specific base pairs (11:24 and 12:23) in the D-stem of the tRNA substrate are involved in interacting with human NSun6. Together, our findings suggest that human NSun6 relies on a delicate network for RNA recognition, which involves both the primary sequence and tertiary structure of tRNA substrates.

Analysis of the Cleavage Mechanism by Protein-Only RNase P Using Precursor tRNA Substrates with Modifications at the Cleavage Site

Ribonuclease P (RNase P) is the enzyme that endonucleolytically removes 5′-precursor sequences from tRNA transcripts in all domains of life. RNase P activities are either ribonucleoprotein (RNP) or protein-only RNase P (PRORP) enzymes, raising the question about the mechanistic strategies utilized by these architecturally different enzyme classes to catalyze the same type of reaction. Here, we analyzed the kinetics and cleavage-site selection by PRORP3 from Arabidopsis thaliana (AtPRORP3) using precursor tRNAs (pre-tRNAs) with individual modifications at the canonical cleavage site, with either Rp- or Sp-phosphorothioate, or 2′-deoxy, 2′-fluoro, 2′-amino, or 2′-O-methyl substitutions. We observed a small but robust rescue effect of Sp-phosphorothioate-modified pre-tRNA in the presence of thiophilic Cd2+ ions, consistent with metal-ion coordination to the (pro-)Sp-oxygen during catalysis. Sp-phosphorothioate, 2′-deoxy, 2′-amino, and 2′-O-methyl modification redirected the cleavage mainly to the next unmodified phosphodiester in the 5′-direction. Our findings are in line with the 2′-OH substituent at nucleotide −1 being involved in an H-bonding acceptor function. In contrast to bacterial RNase P, AtPRORP3 was found to be able to utilize the canonical and upstream cleavage site with similar efficiency (corresponding to reduced cleavage fidelity), and the two cleavage pathways appear less interdependent than in the bacterial RNA-based system.

Functional Diversity of Cytotoxic tRNase/Immunity Protein Complexes from Burkholderia pseudomallei

Contact-dependent growth inhibition (CDI) is a widespread mechanism of inter-bacterial competition. CDI(+) bacteria deploy large CdiA effector proteins, which carry variable C-terminal toxin domains (CdiA-CT). CDI(+) cells also produce CdiI immunity proteins that specifically neutralize cognate CdiA-CT toxins to prevent auto-inhibition. Here, we present the crystal structure of the CdiA-CT/CdiI(E479) toxin/immunity protein complex from Burkholderia pseudomallei isolate E479. The CdiA-CT(E479) tRNase domain contains a core α/β-fold that is characteristic of PD(D/E)XK superfamily nucleases. Unexpectedly, the closest structural homolog of CdiA-CT(E479) is another CDI toxin domain from B. pseudomallei 1026b. Although unrelated in sequence, the two B. pseudomallei nuclease domains share similar folds and active-site architectures. By contrast, the CdiI(E479) and CdiI(1026b) immunity proteins share no significant sequence or structural homology. CdiA-CT(E479) and CdiA-CT(1026b) are both tRNases; however, each nuclease cleaves tRNA at a distinct position. We used a molecular docking approach to model each toxin bound to tRNA substrate. The resulting models fit into electron density envelopes generated by small-angle x-ray scattering analysis of catalytically inactive toxin domains bound stably to tRNA. CdiA-CT(E479) is the third CDI toxin found to have structural homology to the PD(D/E)XK superfamily. We propose that CDI systems exploit the inherent sequence variability and active-site plasticity of PD(D/E)XK nucleases to generate toxin diversity. These findings raise the possibility that many other uncharacterized CDI toxins may belong to the PD(D/E)XK superfamily.

Unraveling the essential role of CysK in CDI toxin activation

Contact-dependent growth inhibition (CDI) is a widespread mechanism of bacterial competition. CDI(+) bacteria deliver the toxic C-terminal region of contact-dependent inhibition A proteins (CdiA-CT) into neighboring target bacteria and produce CDI immunity proteins (CdiI) to protect against self-inhibition. The CdiA-CT(EC536) deployed by uropathogenic Escherichia coli 536 (EC536) is a bacterial toxin 28 (Ntox28) domain that only exhibits ribonuclease activity when bound to the cysteine biosynthetic enzyme O-acetylserine sulfhydrylase A (CysK). Here, we present crystal structures of the CysK/CdiA-CT(EC536) binary complex and the neutralized ternary complex of CysK/CdiA-CT/CdiI(EC536) CdiA-CT(EC536) inserts its C-terminal Gly-Tyr-Gly-Ile peptide tail into the active-site cleft of CysK to anchor the interaction. Remarkably, E. coli serine O-acetyltransferase uses a similar Gly-Asp-Gly-Ile motif to form the "cysteine synthase" complex with CysK. The cysteine synthase complex is found throughout bacteria, protozoa, and plants, indicating that CdiA-CT(EC536) exploits a highly conserved protein-protein interaction to promote its toxicity. CysK significantly increases CdiA-CT(EC536) thermostability and is required for toxin interaction with tRNA substrates. These observations suggest that CysK stabilizes the toxin fold, thereby organizing the nuclease active site for substrate recognition and catalysis. By contrast, Ntox28 domains from Gram-positive bacteria lack C-terminal Gly-Tyr-Gly-Ile motifs, suggesting that they do not interact with CysK. We show that the Ntox28 domain from Ruminococcus lactaris is significantly more thermostable than CdiA-CT(EC536), and its intrinsic tRNA-binding properties support CysK-independent nuclease activity. The striking differences between related Ntox28 domains suggest that CDI toxins may be under evolutionary pressure to maintain low global stability.

From tRNA to miRNA: RNA‐folding contributes to correct entry into noncoding RNA pathways

Although RNA are synthesized as single-stranded molecules, most of them are characterized by extensive secondary structures. Two RNA that require distinct folding for biogenesis and function are miRNA and tRNA. While miRNA are processed from hairpin-containing precursors, tRNA are folded into characteristic L-shaped structures. In addition, tRNA and their precursors are a rich source of RNA fragments. Recent findings suggest that their production might be determined by structural characteristics of the tRNA substrates. Importantly, correct folding of pre-tRNA is assisted by the Lupus autoantigen La, an RNA chaperone. In this context, La interacts with pre-tRNA to prevent alternative foldings leading to mis-channeling into the miRNA pathway. Thus, RNA chaperones also function as gatekeepers for correct RNA pathway selection.

Structural and functional analyses of the archaeal tRNA m2G/m22G10 methyltransferase aTrm11 provide mechanistic insights into site specificity of a tRNA methyltransferase that contains common RNA-binding modules.

N2-methylguanosine is one of the most universal modified nucleosides required for proper function in transfer RNA (tRNA) molecules. In archaeal tRNA species, a specific S-adenosyl-L-methionine (SAM)-dependent tRNA methyltransferase (MTase), aTrm11, catalyzes formation of N2-methylguanosine and N2,N2-dimethylguanosine at position 10. Here, we report the first X-ray crystal structures of aTrm11 from Thermococcus kodakarensis (Tko), of the apo-form, and of its complex with SAM. The structures show that TkoTrm11 consists of three domains: an N-terminal ferredoxinlike domain (NFLD), THUMP domain and Rossmann-fold MTase (RFM) domain. A linker region connects the THUMP-NFLD and RFM domains. One SAM molecule is bound in the pocket of the RFM domain, suggesting that TkoTrm11 uses a catalytic mechanism similar to that of other tRNA MTases containing an RFM domain. Furthermore, the conformation of NFLD and THUMP domains in TkoTrm11 resembles that of other tRNA-modifying enzymes specifically recognizing the tRNA acceptor stem. Our docking model of TkoTrm11-SAM in complex with tRNA, combined with biochemical analyses and pre-existing evidence, provides insights into the substrate tRNA recognition mechanism: The THUMP domain recognizes a 3′-ACCA end, and the linker region and RFM domain recognize the T-stem, acceptor stem and V-loop of tRNA, thereby causing TkoTrm11 to specifically identify its methylation site.

Crystal Structure of the Wild-Type Human GlyRS Bound with tRNAGly in a Productive Conformation

Aminoacyl-tRNA synthetases are essential components of the protein translational machinery in all living species, among which the human glycyl-tRNA synthetase (hGlyRS) is of great research interest because of its unique species-specific aminoacylation properties and noncanonical roles in the Charcot–Marie–Tooth neurological disease. However, the molecular mechanisms of how the enzyme carries out its classical and alternative functions are not well understood. Here, we report a complex structure of the wild-type hGlyRS bound with tRNAGly at 2.95Å. In the complex, the flexible Whep-TRS domain is visible in one of the subunits of the enzyme dimer, and the tRNA molecule is also completely resolved. At the active site, a glycyl-AMP molecule is synthesized and is waiting for the transfer of the glycyl moiety to occur. This cocrystal structure provides us with new details about the recognition mechanism in the intermediate stage during glycylation, which was not well elucidated in the previous crystal structures where the inhibitor AMPPNP was used for crystallization. More importantly, the structural and biochemical work conducted in the current and previous studies allows us to build a model of the full-length hGlyRS in complex with tRNAGly, which greatly helps us to understand the roles that insertions and the Whep-TRS domain play in the tRNA-binding process. Finally, through structure comparison with other class II aminoacyl-tRNA synthetases bound with their tRNA substrates, we found some commonalities of the aminoacylation mechanism between these enzymes.

Identification of protein interfaces within the multi-aminoacyl-tRNA synthetase complex: the case of lysyl-tRNA synthetase and the scaffold protein p38.

Human cytoplasmic lysyl‐tRNA synthetase (LysRS) is associated within a multi‐aminoacyl‐tRNA synthetase complex (MSC). Within this complex, the p38 component is the scaffold protein that binds the catalytic domain of LysRS via its N‐terminal region. In addition to its translational function when associated to the MSC, LysRS is also recruited in nontranslational roles after dissociation from the MSC. The balance between its MSC‐associated and MSC‐dissociated states is essential to regulate the functions of LysRS in cellular homeostasis. With the aim of understanding the rules that govern association of LysRS in the MSC, we analyzed the protein interfaces between LysRS and the full‐length version of p38, the scaffold protein of the MSC. In a previous study, the cocrystal structure of LysRS with a N‐terminal peptide of p38 was reported [Ofir‐Birin Y et al. (2013) Mol Cell 49, 30–42]. In order to identify amino acid residues involved in interaction of the two proteins, the non‐natural, photo‐cross‐linkable amino acid p‐benzoyl‐l‐phenylalanine (Bpa) was incorporated at 27 discrete positions within the catalytic domain of LysRS. Among the 27 distinct LysRS mutants, only those with Bpa inserted in place of Lys356 or His364 were cross‐linked with p38. Using mass spectrometry, we unambiguously identified the protein interface of the cross‐linked complex and showed that Lys356 and His364 of LysRS interact with the peptide from Pro8 to Arg26 in native p38, in agreement with the published cocrystal structure. This interface, which in LysRS is located on the opposite side of the dimer to the site of interaction with its tRNA substrate, defines the core region of the MSC. The residues identified herein in human LysRS are not conserved in yeast LysRS, an enzyme that does not associate within the MSC, and contrast with the residues proposed to be essential for LysRS:p38 association in the earlier work.

Backbone resonance assignments of the m1A22 tRNA methyltransferase TrmK from Bacillus subtilis.

RNA modification is a post-transcriptional process by which certain nucleotides are altered after their initial incorporation into an RNA chain. Transfer RNAs (tRNAs) is the most heavily modified class of RNA molecules. These modifications expand the chemical and functional diversity of tRNAs and enhance their structural stability. To date, more than 100 modifications have been identified, the majority of which are specific from one domain of life. However, few modifications are extensively present in the three domains of life. Among those, the m(1)A nucleotide, which consists in the methylation at position 1 of the adenine aromatic ring, is found in tRNAs and ribosomal RNAs. In tRNAs, the m(1)A modification occurs at position 9, 14, 22, 57 and 58. The enzyme TrmK catalyzes the m(1)A formation at position 22. Here we report the backbone (1)H, (15)N and (13)C chemical shift assignments of TrmK from Bacillus subtilis obtained by heteronuclear multidimensional NMR spectroscopy as well as its secondary structure in solution as predicted by TALOS+. These assignments of TrmK pave the way for interaction studies with its tRNA substrates.

An RNA methylase caught in the act

Structural Biology RNA methylation is important in RNA function and in antibiotic resistance. The RNA methylase RlmN is a dual-specificity enzyme that can act on ribosomal and transfer RNA (tRNA). RlmN is a radical S -adenosylmethionine (SAM) enzyme, which produces a protein/RNA cross-linked intermediate. Schwalm et al. determined the structure of RlmN cross-linked to a tRNA substrate and found that the enzyme recognizes the overall shape of the tRNA. Then it remodels the anticodon region to access the base that it methylates. The remodeling activity is likely to be key to the enzyme's dual specificity. Science , this issue p. [309][1] [1]: /lookup/doi/10.1126/science.aad5367

Crystallographic capture of a radical S-adenosylmethionine enzyme in the act of modifying tRNA.

RlmN is a dual-specificity RNA methylase that modifies C2 of adenosine 2503 (A2503) in 23S rRNA and C2 of adenosine 37 (A37) in several Escherichia coli transfer RNAs (tRNAs). A related methylase, Cfr, modifies C8 of A2503 via a similar mechanism, conferring resistance to multiple classes of antibiotics. Here, we report the x-ray structure of a key intermediate in the RlmN reaction, in which a Cys(118)→Ala variant of the protein is cross-linked to a tRNA(Glu)substrate through the terminal methylene carbon of a formerly methylcysteinyl residue and C2 of A37. RlmN contacts the entire length of tRNA(Glu), accessing A37 by using an induced-fit strategy that completely unfolds the tRNA anticodon stem-loop, which is likely critical for recognition of both tRNA and ribosomal RNA substrates.