Crystal structures of an unmodified bacterial tRNA reveal intrinsic structural flexibility and plasticity as general properties of unbound tRNAs.

Ubiquitous across all domains of life, tRNAs constitute an essential component of cellular physiology, carry out an indispensable role in protein synthesis, and have been historically the subject of a wide range of biochemical and biophysical studies as prototypical folded RNA molecules. Although conformational flexibility is a well-established characteristic of tRNA structure, it is typically regarded as an adaptive property exhibited in response to an inducing event, such as the binding of a tRNA synthetase or the accommodation of an aminoacyl-tRNA into the ribosome. In this study, we present crystallographic data of a tRNA molecule to expand on this paradigm by showing that structural flexibility and plasticity are intrinsic properties of tRNAs, apparent even in the absence of other factors. Based on two closely related conformations observed within the same crystal, we posit that unbound tRNAs by themselves are flexible and dynamic molecules. Furthermore, we demonstrate that the formation of the T-loop conformation by the tRNA TΨC stem-loop, a well-characterized and classic RNA structural motif, is possible even in the absence of important interactions observed in fully folded tRNAs.

Ensemble cryo-EM elucidates the mechanism of translation fidelity.

Gene translation depends on accurate decoding of mRNA, the structural mechanism of which remains poorly understood. Ribosomes decode mRNA codons by selecting cognate aminoacyl-tRNAs delivered by elongation factor Tu (EF-Tu). Here we present high-resolution structural ensembles of ribosomes with cognate or near-cognate aminoacyl-tRNAs delivered by EF-Tu. Both cognate and near-cognate tRNA anticodons explore the aminoacyl-tRNA-binding site (A site) of an open 30S subunit, while inactive EF-Tu is separated from the 50S subunit. A transient conformation of decoding-centre nucleotide G530 stabilizes the cognate codon-anticodon helix, initiating step-wise 'latching' of the decoding centre. The resulting closure of the 30S subunit docks EF-Tu at the sarcin-ricin loop of the 50S subunit, activating EF-Tu for GTP hydrolysis and enabling accommodation of the aminoacyl-tRNA. By contrast, near-cognate complexes fail to induce the G530 latch, thus favouring open 30S pre-accommodation intermediates with inactive EF-Tu. This work reveals long-sought structural differences between the pre-accommodation of cognate and near-cognate tRNAs that elucidate the mechanism of accurate decoding.

tmRNA.SmpB complex mimics native aminoacyl-tRNAs in the A site of stalled ribosomes.

Bacterial ribosomes stalled on faulty, often truncated, mRNAs lacking stop codons are rescued by trans-translation. It relies on an RNA molecule (tmRNA) capable of replacing the faulty mRNA with its own open reading frame (ORF). Translation of tmRNA ORF results in the tagging of faulty protein for degradation and its release from the ribosome. We used single-particle cryo-electron microscopy to visualize tmRNA together with its helper protein SmpB on the 70S Escherichia coli ribosome in states subsequent to GTP hydrolysis on elongation factor Tu (EF-Tu). Three-dimensional reconstruction and heterogeneity analysis resulted in a 15A resolution structure of the tmRNA.SmpB complex accommodated in the A site of the ribosome, which shows that SmpB mimics the anticodon- and D-stem of native tRNAs missing in the tRNA-like domain of tmRNA. We conclude that the tmRNA.SmpB complex accommodates in the ribosomal A site very much like an aminoacyl-tRNA during protein elongation.

Incorporation of aminoacyl-tRNA into the ribosome as seen by cryo-electron microscopy.

Aminoacyl-tRNAs (aa-tRNAs) are delivered to the ribosome as part of the ternary complex of aa-tRNA, elongation factor Tu (EF-Tu) and GTP. Here, we present a cryo-electron microscopy (cryo-EM) study, at a resolution of approximately 9 A, showing that during the incorporation of the aa-tRNA into the 70S ribosome of Escherichia coli, the flexibility of aa-tRNA allows the initial codon recognition and its accommodation into the ribosomal A site. In addition, a conformational change observed in the GTPase-associated center (GAC) of the ribosomal 50S subunit may provide the mechanism by which the ribosome promotes a relative movement of the aa-tRNA with respect to EF-Tu. This relative rearrangement seems to facilitate codon recognition by the incoming aa-tRNA, and to provide the codon-anticodon recognition-dependent signal for the GTPase activity of EF-Tu. From these new findings we propose a mechanism that can explain the sequence of events during the decoding of mRNA on the ribosome.

Cryo-EM reveals an active role for aminoacyl-tRNA in the accommodation process.

During the elongation cycle of protein biosynthesis, the specific amino acid coded for by the mRNA is delivered by a complex that is comprised of the cognate aminoacyl-tRNA, elongation factor Tu and GTP. As this ternary complex binds to the ribosome, the anticodon end of the tRNA reaches the decoding center in the 30S subunit. Here we present the cryo- electron microscopy (EM) study of an Escherichia coli 70S ribosome-bound ternary complex stalled with an antibiotic, kirromycin. In the cryo-EM map the anticodon arm of the tRNA presents a new conformation that appears to facilitate the initial codon-anticodon interaction. Furthermore, the elbow region of the tRNA is seen to contact the GTPase-associated center on the 50S subunit of the ribosome, suggesting an active role of the tRNA in the transmission of the signal prompting the GTP hydrolysis upon codon recognition.

Evidence for aminoacylation-induced conformational changes in human mitochondrial tRNAs.

Analysis by acid polyacrylamide/urea gel electrophoresis of 14 individual mitochondrial tRNAs (mt-tRNAs) from human cells has revealed a variable decrease in mobility of the aminoacylated relative to the nonacylated form, with the degree of separation of the two forms not being correlated with the mass, polar character, or charge of the amino acid. Separation of the charged and uncharged species has been found to be independent of tRNA denaturation, being observed also in the absence of urea. In another approach, electrophoresis through a perpendicular denaturing gradient gel of several individual mt-tRNAs has shown a progressive unfolding of the tRNA with increasing denaturant concentration, which is consistent with an initial disruption of tertiary interactions, followed by the sequential melting of the four stems of the cloverleaf structure. A detailed analysis of the unfolding process of charged and uncharged tRNALys and tRNALeu(UUR) has revealed that the separation of the two forms of these tRNAs persisted throughout the almost entire range of denaturant concentrations used and was lost upon denaturation of the last helical domain(s), which most likely included the amino acid acceptor stem. These observations strongly suggest that the electrophoretic retardation of the charged species reflects an aminoacylation-induced conformational change of the 3'-end of these mt-tRNAs, with possible significant implications in connection with the known role of the acceptor end in tRNA interactions with the ribosomal peptidyl transferase center and the elongation factor Tu.

Chemically synthesised human immunodeficiency virus P7 nucleocapsid protein can self-assemble into particles and binds to a specific site on the tRNA(Lys,3) primer.

The zinc-bound form of the human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein, p7, aggregates into particles visible by electron microscopy. The HIV primer tRNA(Lys,3) forms similar high molecular weight complexes with p7 that are also detected by gel mobility shift assays. RNA oligonucleotides of the three stem-loop structures in tRNA(Lys,3) were assayed for the competitive inhibition of p7-tRNA(Lys,3) binding by the intensities of free tRNA(Lys,3) bands on native gels. This reveals that the p7 binds specifically to the central domain of tRNA(Lys,3) where the D and T psi C loops come together, but not the anticodon stem-loop.

Visualization of a ternary complex of the Escherichia coli Phe-tRNA(Phe) and Tu.GTP from Thermus thermophilus by scanning transmission electron microscopy.

Scanning transmission electron microscopy (STEM) was used to visualize formation of a ternary complex between the T. thermophilus elongation factor (EF) Tu.GTP and the Escherichia coli Phe-tRNA(Phe) labeled with an undecagold (Au11) cluster at minor nucleotide 3-(3-amino-3-carboxypropyl) uridine at position 47. The ternary complex was further characterized by the molecular mass and radius of gyration calculated from the mass distribution within the individual particles. Under conditions used for STEM imaging, the ternary complex is formed between Au11-labeled Phe-tRNA(Phe) and Tu.GTP in a yield up to 25%. The stoichiometry of EF-Tu.GTP to aminoacyl-tRNA (aa-tRNA) in the EF-Tu.GTP.aa-tRNA complex is 1:1, in agreement with the established view of the protein biosynthesis mechanism. The ternary complex is also formed, although to a lower extent, with GTP analogues (GMPPCP and GMPPNP, respectively), but not with Tu.GDP and nonaminoacylated tRNA(Phe) with Tu.GTP.

Class II aminoacyl transfer RNA synthetases: crystal structure of yeast aspartyl-tRNA synthetase complexed with tRNA(Asp).

The crystal structure of the binary complex tRNA(Asp)-aspartyl tRNA synthetase from yeast was solved with the use of multiple isomorphous replacement to 3 angstrom resolution. The dimeric synthetase, a member of class II aminoacyl tRNA synthetases (aaRS's) exhibits the characteristic signature motifs conserved in eight aaRS's. These three sequence motifs are contained in the catalytic site domain, built around an antiparallel beta sheet, and flanked by three alpha helices that form the pocket in which adenosine triphosphate (ATP) and the CCA end of tRNA bind. The tRNA(Asp) molecule approaches the synthetase from the variable loop side. The two major contact areas are with the acceptor end and the anticodon stem and loop. In both sites the protein interacts with the tRNA from the major groove side. The correlation between aaRS class II and the initial site of aminoacylation at 3'-OH can be explained by the structure. The molecular association leads to the following features: (i) the backbone of the GCCA single-stranded portion of the acceptor end exhibits a regular helical conformation; (ii) the loop between residues 320 and 342 in motif 2 interacts with the acceptor stem in the major groove and is in contact with the discriminator base G and the first base pair UA; and (iii) the anticodon loop undergoes a large conformational change in order to bind the protein. The conformation of the tRNA molecule in the complex is dictated more by the interaction with the protein than by its own sequence.

A stereochemical model of the transpeptidation complex.

Molecular models are proposed to describe the relative arrangement of aminoacyl and peptidyl tRNAs when bound to their respective A and P sites on the ribosome. The crystallographically determined structures of tRNAasp and tRNAphe have served as the models for these bound structures, while the imposed steric constraints for the model complexes were based on the results of published experimental data. The constructed models satisfy the stereochemical requirements needed for codon-anticodon interaction and for peptide bond formation. In this paper, the results of the complex containing tRNAphe as the A and P site bound transfer RNAs, is compared to a similarly constructed model which uses tRNAasp as the ribosome-bound transfer RNAs. The models have the following three major features: 1) the aminoacyl and peptidyl transfer RNAs assume an angle of approximately 45 degrees relative to each other; 2) in providing the proper stereochemistry for peptide bond condensation, a significant kink must be present in the messenger RNA between the A site and P site codons; and 3) a comparison of the two model complexes indicates that structural variations between the tRNAs or any allosteric transitions of the transfer RNAs associated with codon-anticodon recognition may be accommodated in the model by way of freedom of rotation about the phosphate backbone bonds in the mRNA between consecutive codons.

The nucleotide sequence, localization and transcriptional properties of a tRNALeuCUG gene from Drosophila melanogaster.

The nucleotide sequence of a tRNALeuCUG gene from Drosophila melanogaster has been determined and compared with available tRNALeuCUG sequences from other eukaryotes, as well as with the tRNALeuUUG gene of D. melanogaster. The genomic location, determined by in situ hybridization, was found to be at site 66B on chromosome 3L. This localization probably places it within one of the known, but uncharacterized, clusters of tRNA genes in this organism. In addition, the transcriptional behaviour of this tRNALeuCUG gene in various in vitro systems is described and it seems that, although the gene is transcribed in all test systems, the very A + T-rich 5'-flanking sequence of this particular gene may be somewhat inhibitory to transcription in vitro.