Anticodon Stem Loop Research Articles

Pseudouridine synthase 1: a site-specific synthase without strict sequence recognition requirements

Pseudouridine synthase 1 (Pus1p) is an unusual site-specific modification enzyme in that it can modify a number of positions in tRNAs and can recognize several other types of RNA. No consensus recognition sequence or structure has been identified for Pus1p. Human Pus1p was used to determine which structural or sequence elements of human tRNASer are necessary for pseudouridine (Ψ) formation at position 28 in the anticodon stem-loop (ASL). Some point mutations in the ASL stem of tRNASer had significant effects on the levels of modification and compensatory mutation, to reform the base pair, restored a wild-type level of Ψ formation. Deletion analysis showed that the tRNASer TΨC stem-loop was a determinant for modification in the ASL. A mini-substrate composed of the ASL and TΨC stem-loop exhibited significant Ψ formation at position 28 and a number of mutants were tested. Substantial base pairing in the ASL stem (3 out of 5 bp) is required, but the sequence of the TΨC loop is not required for modification. When all nucleotides in the ASL stem other than U28 were changed in a single mutant, but base pairing was retained, a near wild-type level of modification was observed.

Nucleotide modifications and tRNA anticodon–mRNA codon interactions on the ribosome

We have carried out molecular dynamics simulations of the tRNA anticodon and mRNA codon, inside the ribosome, to study the effect of the common tRNA modifications cmo(5)U34 and m(6)A37. In tRNA(Val), these modifications allow all four nucleotides to be successfully read at the wobble position in a codon. Previous data suggest that entropic effects are mainly responsible for the extended reading capabilities, but detailed mechanisms have remained unknown. We have performed a wide range of simulations to elucidate the details of these mechanisms at the atomic level and quantify their effects: extensive free energy perturbation coupled with umbrella sampling, entropy calculations of tRNA (free and bound to the ribosome), and thorough structural analysis of the ribosomal decoding center. No prestructuring effect on the tRNA anticodon stem-loop from the two modifications could be observed, but we identified two mechanisms that may contribute to the expanded decoding capability by the modifications: The further reach of the cmo(5)U34 allows an alternative outer conformation to be formed for the noncognate base pairs, and the modification results in increased contacts between tRNA, mRNA, and the ribosome.

Structural and functional basis for RNA cleavage by Ire1

BackgroundThe unfolded protein response (UPR) controls the protein folding capacity of the endoplasmic reticulum (ER). Central to this signaling pathway is the ER-resident bifunctional transmembrane kinase/endoribonuclease Ire1. The endoribonuclease (RNase) domain of Ire1 initiates a non-conventional mRNA splicing reaction, leading to the production of a transcription factor that controls UPR target genes. The mRNA splicing reaction is an obligatory step of Ire1 signaling, yet its mechanism has remained poorly understood due to the absence of substrate-bound crystal structures of Ire1, the lack of structural similarity between Ire1 and other RNases, and a scarcity of quantitative enzymological data. Here, we experimentally define the active site of Ire1 RNase and quantitatively evaluate the contribution of the key active site residues to catalysis.ResultsThis analysis and two new crystal structures suggest that Ire1 RNase uses histidine H1061 and tyrosine Y1043 as the general acid-general base pair contributing ≥ 7.6 kcal/mol and 1.4 kcal/mol to transition state stabilization, respectively, and asparagine N1057 and arginine R1056 for coordination of the scissile phosphate. Investigation of the stem-loop recognition revealed that additionally to the stem-loops derived from the classic Ire1 substrates HAC1 and Xbp1 mRNA, Ire1 can site-specifically and rapidly cleave anticodon stem-loop (ASL) of unmodified tRNAPhe, extending known substrate specificity of Ire1 RNase.ConclusionsOur data define the catalytic center of Ire1 RNase and suggest a mechanism of RNA cleavage: each RNase monomer apparently contains a separate catalytic apparatus for RNA cleavage, whereas two RNase subunits contribute to RNA stem-loop docking. Conservation of the key residues among Ire1 homologues suggests that the mechanism elucidated here for yeast Ire1 applies to Ire1 in metazoan cells, and to the only known Ire1 homologue RNase L.

Chemical synthesis of an RNA sequence containing 2-thiocytidine (s2C): the DY647 labelled anticodon stem and loop sequence of Staphylococcus aureus tRNAArg (s2C32, mnm5U34, t6A37)

Incorporation of 2-thiocytidine (s2C) into a DY647 labelled 17-mer RNA sequence corresponding to the anticodon stem loop of Staphylococcus aureus tRNAArg has been achieved by phosphoramidite chemistry. Key to the success was the use of dilute iodine for the P(III) to P(V) oxidation step plus the choice of the trimethylsilylethyl (tse) ester as protection for the threonyl carboxyl group of t6A. In trial experiments with N4-benzoyl-2-thiocytidine, the use of dilute t-BuOOH in toluene as an alternative oxidising agent caused substantial desulfurization.

The C-terminal end of the Trypanosoma brucei editing deaminase plays a critical role in tRNA binding

Adenosine to inosine editing at the wobble position allows decoding of multiple codons by a single tRNA. This reaction is catalyzed by adenosine deaminases acting on tRNA (ADATs) and is essential for viability. In bacteria, the anticodon-specific enzyme is a homodimer that recognizes a single tRNA substrate (tRNA(Arg)(ACG)) and can efficiently deaminate short anticodon stem-loop mimics of this tRNA in vitro. The eukaryal enzyme is composed of two nonidentical subunits, ADAT2 and ADAT3, which upon heterodimerization, recognize seven to eight different tRNAs as substrates, depending on the organism, and require a full-length tRNA for activity. Although crystallographic data have provided clues to why the bacterial deaminase can utilize short substrates, residues that provide substrate binding and recognition with the eukaryotic enzymes are not currently known. In the present study, we have used a combination of mutagenesis, binding studies, and kinetic analysis to explore the contribution of individual residues in Trypanosoma brucei ADAT2 (TbADAT2) to tRNA recognition. We show that deletion of the last 10 amino acids at the C terminus of TbADAT2 abolishes tRNA binding. In addition, single alanine replacements of a string of positively charged amino acids (KRKRK) lead to binding defects that correlate with losses in enzyme activity. This region, which we have termed the KR-domain, provides a first glance at key residues involved in tRNA binding by eukaryotic tRNA editing deaminases.

Nucleotide Modifications Change tRNA Dynamics and Base Pairing

Modified and unmodified yeast tRNA(Phe) in solution was simulated to understand the effect of nucleotide modifications on the dynamics of tRNA. High performance computing techniques were employed to obtain a “dynamic picture” at spacial and time resolutions hardly accessible experimentally for these systems. Local flexibility, secondary and tertiary structure, global rearrangements and movements of the whole tRNA were probed by a microsecond of all atom explicit solvent molecular dynamics simulations.The results of our simulations give new insight on experimentally observed biological impact of nucleotide modifications. Amongst other results we find that tRNA modification leads to a decrease in secondary structure and tertiary interactions in the anticodon stem loop. Nucleotides U16 and U17 show different orientations when modified to dihydrouridine. Modification of A58 to 1-methyladenosine causes local rearrangements in the elbow region. Implications of our results on external factor binding are discussed.

The AlkB domain of mammalian ABH8 catalyzes hydroxylation of 5-methoxycarbonylmethyluridine at the wobble position of tRNA.

Family ties: The AlkB family of nonheme iron, α-ketoglutarate (αKG)-dependent dioxygenases is involved in biological processes such as DNA/RNA repair and obesity. The AlkB domain of ABH8 is shown to catalyze the hydroxylation of a modified uridine (mcm5U) into (S)-mchm5U at the wobble position of the anticodon stem–loop in tRNA (see scheme), thereby suggesting that this protein is a tRNA hypermodification enzyme. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

PSRP1 Is Not a Ribosomal Protein, but a Ribosome-binding Factor That Is Recycled by the Ribosome-recycling Factor (RRF) and Elongation Factor G (EF-G)

Plastid-specific ribosomal proteins (PSRPs) have been proposed to play roles in the light-dependent regulation of chloroplast translation. Here we demonstrate that PSRP1 is not a bona fide ribosomal protein, but rather a functional homologue of the Escherichia coli cold-shock protein pY. Three-dimensional Cryo-electron microscopic (Cryo-EM) reconstructions reveal that, like pY, PSRP1 binds within the intersubunit space of the 70S ribosome, at a site overlapping the positions of mRNA and A- and P-site tRNAs. PSRP1 induces conformational changes within ribosomal components that comprise several intersubunit bridges, including bridge B2a, thereby stabilizes the ribosome against dissociation. We find that the presence of PSRP1/pY lowers the binding of tRNA to the ribosome. Furthermore, similarly to tRNAs, PSRP1/pY is recycled from the ribosome by the concerted action of the ribosome-recycling factor (RRF) and elongation factor G (EF-G). These results suggest a novel function for EF-G and RRF in the post-stress return of PSRP1/pY-inactivated ribosomes to the actively translating pool.

Snapshots of Dynamics in Synthesizing N6-Isopentenyladenosine at the tRNA Anticodon

Bacterial and eukaryotic tRNAs that decode codons starting with uridine have a hydrophobically hypermodified adenosine at position 37 (A(37)) adjacent to the 3'-end of the anticodon, which is essential for efficient and highly accurate protein translation by the ribosome. However, it remains unclear as to how the corresponding tRNAs are selected to be modified by alkylation at the correct position of the adenosine base. We have determined a series of crystal structures of bacterial tRNA isopentenyltransferase (MiaA) in apo- and tRNA-bound forms, which completely render snapshots of substrate selections during the modification of RNA. A compact evolutionary inserted domain (herein swinging domain) in MiaA that exhibits as a highly mobile entity moves around the catalytic domain as likely to reach and trap the tRNA substrate. Thereby, MiaA clamps the anticodon stem loop of the tRNA substrate between the catalytic and swinging domains, where the two conserved elongated residues from the swinging domain pinch the two flanking A(36) and A(38) together to squeeze out A(37) into the reaction tunnel. The site-specific isopentenylation of RNA is thus ensured by a characteristic pinch-and-flip mechanism and by a reaction tunnel to confine the substrate selection. Furthermore, combining information from soaking experiments with structural comparisons, we propose a mechanism for the ordered substrate binding of MiaA.

Structure–activity relationships in Kluyveromyces lactis γ-toxin, a eukaryal tRNA anticodon nuclease

tRNA anticodon damage inflicted by secreted ribotoxins such as Kluyveromyces lactis gamma-toxin and bacterial colicins underlies a rudimentary innate immune system that distinguishes self from nonself species. The intracellular expression of gamma-toxin (a 232-amino acid polypeptide) arrests the growth of Saccharomyces cerevisiae by incising a single RNA phosphodiester 3' of the modified wobble base of tRNA(Glu). Fungal gamma-toxin bears no primary structure similarity to any known nuclease and has no plausible homologs in the protein database. To gain insight to gamma-toxin's mechanism, we tested the effects of alanine mutations at 62 basic, acidic, and polar amino acids on ribotoxin activity in vivo. We thereby identified 22 essential residues, including 10 lysines, seven arginines, three glutamates, one cysteine, and one histidine (His209, the only histidine present in gamma-toxin). Structure-activity relations were gleaned from the effects of 44 conservative substitutions. Recombinant tag-free gamma-toxin, a monomeric protein, incised an oligonucleotide corresponding to the anticodon stem-loop of tRNA(Glu) at a single phosphodiester 3' of the wobble uridine. The anticodon nuclease was metal independent. RNA cleavage was abolished by ribose 2'-H and 2'-F modifications of the wobble uridine. Mutating His209 to alanine, glutamine, or asparagine abolished nuclease activity. We propose that gamma-toxin catalyzes an RNase A-like transesterification reaction that relies on His209 and a second nonhistidine side chain as general acid-base catalysts.

Crystallographic studies of ribosomal frameshifting

A recent 2.8 Åcrystal structure of the 70S ribosome from Thermus thermophilus shows many details of how the ribosome interacts with its mRNA and tRNA substrates (1). During translation, specific tRNA‐mRNA combinations can cause frameshifting, deviations from the normal three‐base reading frame. The aim of this work is to gain structural and functional understanding of why these deviations occur and how the ribosome in the normal case prevents them from occurring.Mutated frameshift suppressor tRNAs containing eight‐base anticodon loops can cause a +1 frameshift through decoding of a four‐base codon. The crystal structures of such ASLs (anticodon stem‐loops) bound to their cognate four‐base mRNA codons in the context of the ribosomal A site showed that three bases of the extended anticodon covered four bases of the mRNA (2).When the tRNA balance is altered, tRNA3Ser, that normally decodes AGU and AGC, can cause ‐1 frameshifting at a GCA alanine codon. Nitrocellulose filter binding studies indicate that this occurs through a doublet decoding mechanism.The current status of our crystallographic studies of frameshift‐related ribosomal complexes will be presented.1. Selmer, M et al., Science 313(5795):1935‐1942 (2006)2. Dunham, C.M. et al., RNA 13(6):817‐23 (2007)

GTPase activation of elongation factor EF-Tu by the ribosome during decoding

We have used single-particle reconstruction in cryo-electron microscopy to determine a structure of the Thermus thermophilus ribosome in which the ternary complex of elongation factor Tu (EF-Tu), tRNA and guanine nucleotide has been trapped on the ribosome using the antibiotic kirromycin. This represents the state in the decoding process just after codon recognition by tRNA and the resulting GTP hydrolysis by EF-Tu, but before the release of EF-Tu from the ribosome. Progress in sample purification and image processing made it possible to reach a resolution of 6.4 A. Secondary structure elements in tRNA, EF-Tu and the ribosome, and even GDP and kirromycin, could all be visualized directly. The structure reveals a complex conformational rearrangement of the tRNA in the A/T state and the interactions with the functionally important switch regions of EF-Tu crucial to GTP hydrolysis. Thus, the structure provides insights into the molecular mechanism of signalling codon recognition from the decoding centre of the 30S subunit to the GTPase centre of EF-Tu.

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.

The crystal structure of Pyrococcus abyssi tRNA (uracil-54, C5)-methyltransferase provides insights into its tRNA specificity

The 5-methyluridine is invariably found at position 54 in the TΨC loop of tRNAs of most organisms. In Pyrococcus abyssi, its formation is catalyzed by the S-adenosyl-l-methionine-dependent tRNA (uracil-54, C5)-methyltransferase (PabTrmU54), an enzyme that emerged through an ancient horizontal transfer of an RNA (uracil, C5)-methyltransferase-like gene from bacteria to archaea. The crystal structure of PabTrmU54 in complex with S-adenosyl-l-homocysteine at 1.9 Å resolution shows the protein organized into three domains like Escherichia coli RumA, which catalyzes the same reaction at position 1939 of 23S rRNA. A positively charged groove at the interface between the three domains probably locates part of the tRNA-binding site of PabTrmU54. We show that a mini-tRNA lacking both the D and anticodon stem-loops is recognized by PabTrmU54. These results were used to model yeast tRNAAsp in the PabTrmU54 structure to get further insights into the different RNA specificities of RumA and PabTrmU54. Interestingly, the presence of two flexible loops in the central domain, unique to PabTrmU54, may explain the different substrate selectivities of both enzymes. We also predict that a large TΨC loop conformational change has to occur for the flipping of the target uridine into the PabTrmU54 active site during catalysis.

Modified Uridines with C5-methylene Substituents at the First Position of the tRNA Anticodon Stabilize U·G Wobble Pairing during Decoding

Post-transcriptional modifications at the first (wobble) position of the tRNA anticodon participate in precise decoding of the genetic code. To decode codons that end in a purine (R) (i.e. NNR), tRNAs frequently utilize 5-methyluridine derivatives (xm(5)U) at the wobble position. However, the functional properties of the C5-substituents of xm(5)U in codon recognition remain elusive. We previously found that mitochondrial tRNAs(Leu(UUR)) with pathogenic point mutations isolated from MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) patients lacked the 5-taurinomethyluridine (taum(5)U) modification and caused a decoding defect. Here, we constructed Escherichia coli tRNAs(Leu(UUR)) with or without xm(5)U modifications at the wobble position and measured their decoding activities in an in vitro translation as well as by A-site tRNA binding. In addition, the decoding properties of tRNA(Arg) lacking mnm(5)U modification in a knock-out strain of the modifying enzyme (DeltamnmE) were examined by pulse labeling using reporter constructs with consecutive AGR codons. Our results demonstrate that the xm(5)U modification plays a critical role in decoding NNG codons by stabilizing U.G pairing at the wobble position. Crystal structures of an anticodon stem-loop containing taum(5)U interacting with a UUA or UUG codon at the ribosomal A-site revealed that the taum(5)U.G base pair does not have classical U.G wobble geometry. These structures provide help to explain how the taum(5)U modification enables efficient decoding of UUG codons.

Analysis of genomic tRNA sets from Bacteria, Archaea, and Eukarya points to anticodon–codon hydrogen bonds as a major determinant of tRNA compositional variations

Analysis of 100 complete sets of the cytoplasmic elongator tRNA genes from Bacteria, Archaea, and Eukarya pointed to correspondences between types of anticodon and composition of the rest of the tRNA body. The number of the hydrogen bonds formed between the complementary nucleotides in the anticodon-codon duplex appeared as a major quantitative parameter determining covariations in all three domains of life. Our analysis has supported and advanced the "extended anticodon" concept that is based on the argument that the decoding performance of the anticodon is enhanced by selection of a matching anticodon stem-loop sequence, as reported by Yarus in 1982. In addition to the anticodon stem-loop, we have found covariations between the anticodon nucleotides and the composition of the distant regions of their respective tRNAs that include dihydrouridine (D) and thymidyl (T) stem-loops. The majority of the covariable tRNA positions were found at the regions with the increased dynamic potential--such as stem-loop and stem-stem junctions. The consistent occurrences of the covariations on the multigenomic level suggest that the number and pattern of the hydrogen bonds in the anticodon-codon duplex constitute a major factor in the course of translation that is reflected in the fine-tuning of the tRNA composition and structure.

Kluyveromyces lactis -toxin, a ribonuclease that recognizes the anticodon stem loop of tRNA

Kluyveromyces lactis γ-toxin is a tRNA endonuclease that cleaves Saccharomyces cerevisiae , and between position 34 and position 35. All three substrate tRNAs carry a 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) residue at position 34 (wobble position) of which the mcm5 group is required for efficient cleavage. However, the different cleavage efficiencies of mcm5s2U34-containing tRNAs suggest that additional features of these tRNAs affect cleavage. In the present study, we show that a stable anticodon stem and the anticodon loop are the minimal requirements for cleavage by γ-toxin. A synthetic minihelix RNA corresponding to the anticodon stem loop (ASL) of the natural substrate is cleaved at the same position as the natural substrate. In , the nucleotides U34U35C36A37C38 are required for optimal γ-toxin cleavage, whereas a purine at position 32 or a G in position 33 dramatically reduces the cleavage of the ASL. Comparing modified and partially modified forms of E. coli and yeast reinforced the strong stimulatory effects of the mcm5 group, revealed a weak positive effect of the s2 group and a negative effect of the bacterial 5-methylaminomethyl (mnm5) group. The data underscore the high specificity of this yeast tRNA toxin.

Transfer RNA in the hybrid P/E state: correlating molecular dynamics simulations with cryo-EM data.

Transfer RNA (tRNA) transiently occupies the hybrid P/E state (P/E-tRNA) when mRNA-tRNA are translocated in the ribosome. In this study, we characterize the structure of P/E-tRNA and its interactions with the ribosome by correlating the results from molecular dynamics simulations on free tRNA with the cryo-EM map of P/E-tRNA. In our approach, we show that the cryo-EM map may be interpreted as a conformational average. Along the molecular dynamics trajectories (44 ns, 18 ns, and 18 ns), some of the snapshots prove to be quite close to the observed density. In a representative structure, the CCA (3') arm is uniquely twisted, and the anticodon stem loop is kinked at the junctions to both the anticodon loop and the D stem. In addition, the map shows that the P/E-tRNA is no longer bound to helix H69 of 23S rRNA and is flexible, and the conformations of helices H68 and h44 of 16S rRNA differ from those in the x-ray structure. Thus, our study presents structural and dynamic information on the P/E-tRNA and characterizes its interactions with the translocating ribosome.

Parallel dimerization of a PrrC-anticodon nuclease region implicated in tRNALys recognition

The optional Escherichia coli restriction tRNase PrrC represents a family of potential antiviral devices widespread among bacteria. PrrC comprises a functional C-domain of unknown structure and regulatory ABC/ATPase-like N-domain. The possible involvement of a C-domain sequence in tRNALys recognition was investigated using a matching end-protected 11-meric peptide. This mimic, termed here LARP (Lys-anticodon recognizing peptide) UV-cross-linked tRNALys anticodon stem-loop (ASL) analogs and inhibited their PrrC-catalyzed cleavage. Trimming LARP or introducing in it inactivating PrrC missense mutations impaired these activities. LARP appeared to mimic its matching protein sequence in ability to dimerize in parallel, as inferred from the following results. First, tethering Cys to the amino- or carboxy-end of LARP dramatically enhanced the ASL-cross-linking and PrrC-inhibiting activities under suitable redox conditions. Second, Cys-substitutions in a C-domain region containing the sequence corresponding to LARP elicited specific intersubunit cross-links. The parallel dimerization of PrrC's C-domains and expected head-to-tail dimerization of its N-domains further suggest that the NTPase and tRNALys-binding sites of PrrC arise during distinct assembly stages of its dimer of dimers form.

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.