tRNA-mRNA Complex Research Articles

Improving RNA Crystal Diffraction Quality by Postcrystallization Treatment.

The crystallization and structural determination of large RNAs and their complexes remain major bottlenecks in the mechanistic analysis of cellular and viral RNAs. Here, we describe a protocol that combines postcrystallization dehydration and ion replacement that dramatically improved the diffraction quality of crystals of a large gene-regulatory tRNA-mRNA complex. Through this method, the resolution limit of X-ray data extended from 8.5 to 3.2Å, enabling structure determination. Although this protocol was developed for a particular RNA complex, the general importance of solvent and counterions in nucleic acid structure may render it generally useful for crystallographic analysis of other RNAs.

Elucidating the roles of naturally occurring silent mutations in Polycystic Ovary Syndrome (PCOS)

Polycystic ovary syndrome (PCOS) is a complex genetic disorder, which often causes infertility in women. Most of the PCOS-related genes are also linked with metabolic diseases, inflammatory responses, or certain types of cancer. GWAS studies connect single nucleotide polymorphisms (SNPs) with these diseases, emphasizing their critical role in understanding the impacts of SNPs that exchange nucleotides. However, their response to changes in protein conformation and corresponding functional characteristics remains ambiguous. The present study provides evidence that elucidates for the role of specific SNPs known as silent mutations by studying impacts on INSR (insulin receptor), FST (follistatin), and AR (androgen receptor) genes, all strongly associated with PCOS pathogenesis. Although silent mutations do not change the amino acid type, their effects on protein expression may cause disease progression. To assess the potential impacts of silent mutations, we first calculated the anti-codon availabilities through RFMapp. To avoid the controversial issues regarding anti-codon stability for certain SNPs, MFE (minimum free energy) was calculated as a measure of the thermodynamic stability of newly formed mRNA structures to provide more information. For rs2059806 and rs1799817 located in the INSR gene, we reported depletion in anti-codon availabilities of newly existed codons. For rs6152 and rs2059806 cases, we calculated both increase and decrease in mRNA stabilities, which were noted as instabilities of the tRNA-mRNA complex. With the results obtained, we referred to the case of codon optimality for providing a plausible explanation between the existence of these SNPs and PCOS. Herein, the effects of silent mutations on complex PCOS diseases have been demonstrated.

Step back for seminal translation.

Translation elongation entails a one-codon movement of the mRNA–tRNA complex along the mRNA and is catalyzed by the forward translocase EF-G. The structurally related back-translocase EF4 catalyzes movement in the opposite direction when the ribosome stalls, but its physiological role in mammals had been unknown. Genetic ablation of EF4 in mice is now found to cause testis-specific mitochondrial deficiency and impaired spermatogenesis.

Modeling Ribosomal Translocation Facilitated by Peptidyl Transferase Antibiotics

The peptidyl transferase antibiotic sparsomycin can induce efficient ribosomal translocation in the absence of EF-G and GTP. However, how the antibiotic facilitates the translocation is unclear. Here, two models of antibiotic-induced translocation are considered, in which the interaction of the antibiotic with the peptidyl-tRNA in the A/P state, besides behaving as a pawl in the “Brownian ratchet”, also has an effect of compromising the interaction of the 30S subunit with the mRNA-tRNA complex (Model I) or facilitating the forward 30S head rotation which results in the ribosomal unlocking (Model II). It is shown that although the results obtained with Model I can explain the available experimental data on the translocation through the single-stranded mRNA, they are inconsistent with the experimental data on the translocation through the mRNA duplex; by contrast, the results obtained with Model II can explain quantitatively all of the available experimental data. With model II it is further shown that the efficient translocation induced by the binding of sparsomycin results mainly from the facilitation of the forward 30S head rotation, whereas the effect as the pawl plays only a little role. In addition, it is noted that the mRNA movement is brought about by the reverse intersubunit rotation rather than driven directly by the forward 30S head rotation, and the mechanism of antibiotic-induced translocation is similar to that of EF-G-catalyzed translocation.

Post-crystallization Improvement of RNA Crystal Diffraction Quality.

The crystallization and structural determination of large RNAs and their complexes remain major bottlenecks in the mechanistic analysis of cellular and viral RNAs. Here, we describe a protocol that combines post-crystallization dehydration and ion replacement that dramatically improved the diffraction quality of crystals of a large gene-regulatory tRNA-mRNA complex. Through this method, the resolution limit of X-ray data extended from 8.5 to 3.2 Å, enabling structure determination. Although this protocol was developed for a particular RNA complex, the general importance of solvent and counterions in nucleic acid structure may render it generally useful for crystallographic analysis of other RNAs.

Post-crystallization Improvement of RNA Crystals by Synergistic Ion Exchange and Dehydration

Compared to the recent dramatic growth in the numbers of genome-wide and functional studies of complex non-coding RNAs, mechanistic and structural analyses have lagged behind. A major technical bottleneck in the structural determination of large RNAs and their complexes is preparation of diffracting crystals. Empirically, a vast majority of such RNA crystals fail to diffract X-rays to usable resolution (~4 Å) due to their inherent disorder and non-specific packing within the crystals. Here, we present a protocol that combines post-crystallization cation replacement and dehydration that dramatically improved the diffraction quality of crystals of a large gene-regulatory mRNA-tRNA complex. This procedure not only extended the resolution limit of X-ray data from 8.5 to 3.2 Å, but also significantly improved the quality of the data, enabling de novo phasing and structure determination. Because it exploits the general importance of counterions and solvation in RNA structure, this procedure may prove broadly useful in the crystallographic analyses of other large non-coding RNAs.

Modification interference analysis of the ribosome.

RNAs are versatile molecules involved in myriad functions in the cell. To understand how a RNA molecule functions in the cell it is important to identify the nucleotides in the RNA molecule that are important for its structure and function. There are several biochemical methods such as footprinting, cross-linking, and modification interference analysis that can be used to study RNA-RNA and RNA-protein interactions. Ribosome is a classical example of a RNA-protein complex that has been extensively studied using these methods. Here, we describe a modification interference method that was used to identify bases in 16S rRNA that are important for the translocation of the mRNA-tRNA complex by the ribosome.

Dynamics of Forward and Backward Translocation of mRNA in the Ribosome

Translocation of the mRNA-tRNA complex in the ribosome, which is catalyzed by elongation factor EF-G, is one of critical steps in the elongation cycle of protein synthesis. Besides this conventional forward translocation, the backward translocation can also occur, which can be catalyzed by elongation factor LepA. However, the molecular mechanism of the translocation remains elusive. To understand the mechanism, here we study theoretically the dynamics of the forward translocation under various nucleotide states of EF-G and the backward translocation in the absence of and in the presence of LepA. We present a consistent explanation of spontaneous forward translocations in the absence of EF-G, the EF-G-catalyzed forward translocations in the presence of a non-hydrolysable GTP analogue and in the presence of GTP, and the spontaneous and LepA-catalyzed backward translocation. The theoretical results provide quantitative explanations of a lot of different, independent experimental data, and also provide testable predictions.

ADP-ribosylation of Translation Elongation Factor 2 by Diphtheria Toxin in Yeast Inhibits Translation and Cell Separation

Eukaryotic translation elongation factor 2 (eEF2) facilitates the movement of the peptidyl tRNA-mRNA complex from the A site of the ribosome to the P site during protein synthesis. ADP-ribosylation (ADP(R)) of eEF2 by bacterial toxins on a unique diphthamide residue inhibits its translocation activity, but the mechanism is unclear. We have employed a hormone-inducible diphtheria toxin (DT) expression system in Saccharomyces cerevisiae which allows for the rapid induction of ADP(R)-eEF2 to examine the effects of DT in vivo. ADP(R) of eEF2 resulted in a decrease in total protein synthesis consistent with a defect in translation elongation. Association of eEF2 with polyribosomes, however, was unchanged upon expression of DT. Upon prolonged exposure to DT, cells with an abnormal morphology and increased DNA content accumulated. This observation was specific to DT expression and was not observed when translation elongation was inhibited by other methods. Examination of these cells by electron microscopy indicated a defect in cell separation following mitosis. These results suggest that expression of proteins late in the cell cycle is particularly sensitive to inhibition by ADP(R)-eEF2.

Translocation dynamics of tRNA–mRNA in the ribosome

Translocation of tRNA–mRNA complex in the ribosome is an essential step in the elongation cycle of protein synthesis. However, some important issues concerning the molecular mechanism of the tRNA–mRNA translocation catalyzed by EF-G.GTP or by EF-G.GDPNP remain controversial. For example, can EF-G.GTP selectively bind to the hybrid pretranslocation state or bind to both the non-rotated pretranslocation and the hybrid pretranslocation states? Does the greater potency of EF-G in the presence of GTP rather than GDPNP in facilitating translocation derive from the effects on transition from the classical non-rotated to hybrid state (the first step of the translocation) or on transition from the hybrid to posttranslocation state (the second step)? Here, based on our proposed model, we study theoretically the dynamics of the tRNA–mRNA translocation through the ribosome catalyzed by EF-G.GTP and by EF-G.GDPNP. By comparing our theoretical results with the available experimental data, we show that EF-G.GTP can also bind to the classical non-rotated pretranslocation state and the greater potency of GTP hydrolysis in facilitating translocation of tRNA–mRNA complex derives from its effects on the second step of the translocation process.

Bases in 16S rRNA Important for Subunit Association, tRNA Binding, and Translocation

Ribosomes are the cellular machinery responsible for protein synthesis. A well-orchestrated step in the elongation cycle of protein synthesis is the precise translocation of the tRNA-mRNA complex within the ribosome. Here we report the application of a new in vitro modification-interference method for the identification of bases in 16S rRNA that are essential for translocation. Our results suggest that conserved bases U56, U723, A1306, A1319, and A1468 in 16S rRNA are important for translocation. These five bases were deleted or mutated so their role in translation could be studied. Depending on the type of mutation, we observed inhibition of growth rate, subunit association, tRNA binding, and/or translocation. Interestingly, deletion of U56 or A1319 or mutation of A1319 to C showed a lethal phenotype and were defective in protein synthesis in vitro. Further analysis showed that deletion of U56 or A1319 caused defects in 30S subunit assembly, subunit association, and tRNA binding. In contrast, the A1319C mutation showed no defects in subunit association; however, the extent of tRNA binding and translocation was significantly reduced. These results show that conserved bases located as far as 100 A from the tRNA binding sites can be important for translation.

Visualization of the Hybrid State of tRNA Binding Promoted by Spontaneous Ratcheting of the Ribosome

A crucial step in translation is the translocation of tRNAs through the ribosome. In the transition from one canonical site to the other, the tRNAs acquire intermediate configurations, so-called hybrid states. At this stage, the small subunit is rotated with respect to the large subunit, and the anticodon stem loops reside in the A and P sites of the small subunit, while the acceptor ends interact with the P and E sites of the large subunit. In this work, by means of cryo-EM and particle classification procedures, we visualize the hybrid state of both A/P and P/E tRNAs in an authentic factor-free ribosome complex during translocation. In addition, we show how the repositioning of the tRNAs goes hand in hand with the change in the interplay between S13, L1 stalk, L5, H68, H69, and H38 that is caused by the ratcheting of the small subunit.

Crystal Structure of the Bacterial Ribosomal Decoding Site Complexed with a Synthetic Doubly Functionalized Paromomycin Derivative: a New Specific Binding Mode to an A‐Minor Motif Enhances in vitro Antibacterial Activity

The crystal structure of the complex between oligonucleotide containing the bacterial ribosomal decoding site (A site) and the synthetic paromomycin analogue 1, which contains the gamma-amino-alpha-hydroxybutyryl (L-haba) group at position N1 of ring II (2-DOS ring), and an ether chain with an O-phenethylaminoethyl group at position C2'' of ring III, is reported. Interestingly, next to the paromomycin analogue 1 specifically bound to the A site, a second molecule of 1 with a different conformation is observed at the crystal packing interface which mimics the A-minor interaction between two bulged-out adenines from the A site and the codon-anticodon stem of the mRNA-tRNA complex. Improved antibacterial activity supports the conclusion that analogue 1 might affect protein synthesis on the ribosome in two different ways: 1) specific binding to the A site forces maintenance of the "on" state with two bulged out adenines, and 2) a new binding mode of 1 to an A-minor motif which stabilizes complex formation between the ribosome and the mRNA-tRNA complex regardless of whether the codon-anticodon stem is of the cognate or near-cognate type.

Structures of modified eEF2·80S ribosome complexes reveal the role of GTP hydrolysis in translocation

On the basis of kinetic data on ribosome protein synthesis, the mechanical energy for translocation of the mRNA-tRNA complex is thought to be provided by GTP hydrolysis of an elongation factor (eEF2 in eukaryotes, EF-G in bacteria). We have obtained cryo-EM reconstructions of eukaryotic ribosomes complexed with ADP-ribosylated eEF2 (ADPR-eEF2), before and after GTP hydrolysis, providing a structural basis for analyzing the GTPase-coupled mechanism of translocation. Using the ADP-ribosyl group as a distinct marker, we observe conformational changes of ADPR-eEF2 that are due strictly to GTP hydrolysis. These movements are likely representative of native eEF2 motions in a physiological context and are sufficient to uncouple the mRNA-tRNA complex from two universally conserved bases in the ribosomal decoding center (A1492 and A1493 in Escherichia coli) during translocation. Interpretation of these data provides a detailed two-step model of translocation that begins with the eEF2/EF-G binding-induced ratcheting motion of the small ribosomal subunit. GTP hydrolysis then uncouples the mRNA-tRNA complex from the decoding center so translocation of the mRNA-tRNA moiety may be completed by a head rotation of the small subunit.

Translocation of a tRNA with an Extended Anticodon Through the Ribosome

Coordinated translocation of the tRNA–mRNA complex by the ribosome occurs in a precise, stepwise movement corresponding to a distance of three nucleotides along the mRNA. Frameshift suppressor tRNAs generally contain an extra nucleotide in the anticodon loop and they subvert the normal mechanisms used by the ribosome for frame maintenance. The mechanism by which suppressor tRNAs traverse the ribosome during translocation is poorly understood. Here, we demonstrate translocation of a tRNA by four nucleotides from the A site to the P site, and from the P site to the E site. We show that translocation of a punctuated mRNA is possible with an extra, unpaired nucleotide between codons. Interestingly, the NMR structure of the four nucleotide anticodon stem–loop reveals a conformation different from the canonical tRNA structure. Flexibility within the loop may allow conformational adjustment upon A site binding and for interacting with the four nucleotide codon in order to shift the mRNA reading frame.

Ribose 2′-hydroxyl groups in the 5′ strand of the acceptor arm of P-site tRNA are not essential for EF-G catalyzed translocation

The coupled movement of tRNA-mRNA complex through the ribosome is a fundamental step during the protein elongation process. We demonstrate that the ribosome will translocate a P-site-bound tRNA(Met) with a break in the phosphodiester backbone between positions 17 and 18 in the D-loop. Crystallographic data showed that the acceptor arms of P- and E-site tRNA interact extensively with the ribosomal large subunit. Therefore, we used this fragmented P-site-bound tRNA(Met) to investigate the contributions of single 2'-hydroxyl groups in the 5' strand of the acceptor arm for translocation into the ribosomal E-site. EF-G-dependent translocation of the tRNAs was monitored using a toeprinting assay and a fluorescence-based rapid kinetic method. Surprisingly, our results show that none of the 2'-hydroxyl groups in the 5' strand of the acceptor arm of P-site-bound tRNA(Met) between positions 1-17 play a critical role during translocation. This suggests that either these 2'-hydroxyl groups are not important for translocation or they are redundant and the three-dimensional shape of the P-site tRNA is more important for translocation.

GTPases of the Translation Apparatus

Protein biosynthesis is a complex biochemical process involving a number of stages at which different translation factors specifically interact with ribosome. Some of these factors belong to GTP-binding proteins, or G-proteins. Due to their functioning, GTP is hydrolyzed to yield GDP and the inorganic phosphate ion Pi. Interaction with ribosome enhances GTPase activity of translation factors; i.e., ribosome plays a role of GTPase-activating protein (GAP). GTPases involved in translation interact with ribosome at every stage of protein biosynthesis. Initiation factor 2 (IF2) catalyzes initiator tRNA binding to the ribosome P site and subsequent binding of the 50S subunit to the initiation complex of the 30S subunit. Elongation factor Tu (EF-Tu) controls aminoacyl-tRNA delivery to the ribosome A site, while elongation factor G (EF-G) catalyzes translocation of the mRNA-tRNA complex by one codon on the ribosome. Release factor 3 (RF3) catalyzes the release of termination factors 1 or 2 (RF1 or RF2) from the ribosomal complex after completion of protein synthesis and peptidyl-tRNA hydrolysis. The functional properties of translational GTPases as related to other G-proteins, the putative mechanism of GTP hydrolysis, structural features, and the functional cycles of translational GTPases are considered.

Rapid Kinetic Analysis of EF-G-dependent mRNA Translocation in the Ribosome

Precise and coordinated movement of the tRNA–mRNA complex within the ribosome is a fundamental step during protein biosynthesis. The molecular mechanism for this process is still poorly understood. Here we describe a new sensitive method for monitoring elongation factor G-dependent translocation of the mRNA in the ribosome. In this method, the fluorescent probe pyrene is covalently attached to the 3′ end of a short mRNA sequence at position +9. Translocation of the mRNA by one codon results in a significant decrease in the fluorescence emission of pyrene and can be used to directly monitor mRNA movement using rapid kinetic methods. Importantly, this method offers the flexibility of using any tRNA or tRNA analog in order to elucidate the molecular mechanism of translocation. Our results show that the mRNA is translocated at the same rate as the tRNAs, which is consistent with the view that the movement of the tRNAs and the mRNA are coupled in the ribosome. Furthermore, an anticodon stem-loop analog of tRNA is translocated from the ribosomal A site at a rate constant that is 350-fold lower than peptidyl tRNA, indicating that the D stem, T stem and acceptor stem of A site tRNA contribute significantly to the rate of translocation.

After the ribosome structure: how does translocation work?

Structures of the ribosomal large and small subunits have been solved to atomic resolution by X-ray crystallography. These structures provide a new foundation to address the complex process of protein biosynthesis by the ribosome. Translocation of the tRNA-mRNA complex is one of the most fascinating tasks performed by the ribosome. The impact of the crystal structures in understanding the molecular mechanism of translocation is highlighted in this review.

Universally Conserved Interactions between the Ribosome and the Anticodon Stem-Loop of A Site tRNA Important for Translocation

The iterative movement of the tRNA-mRNA complex through the ribosome is a hallmark of the elongation phase of protein synthesis. We used synthetic anticodon stem-loop analogs (ASL) of tRNA Phe to systematically identify ribose 2′-hydroxyl groups that are essential for binding and translocation from the ribosomal A site. Our results show that 2′-hydroxyl groups at positions 33, 35, and 36 in the A site ASL are important for translocation. Consistent with the view that the molecular basis of translocation may be similar in all organisms, the 2′-hydroxyl groups at positions 35 and 36 in the ASL interact with universally conserved bases G530 and A1493, respectively, in 16S rRNA. Furthermore, these interactions are also essential for the decoding process, indicating a functional relationship between decoding and translocation.