Functional importance of Ψ38 and Ψ39 in distinct tRNAs,amplified for tRNAGln(UUG) by unexpected temperature sensitivity of the s2U modification in yeast

The numerous modifications of tRNA play central roles in controlling tRNA structure and translation. Modifications in and around the anticodon loop often have critical roles in decoding mRNA and in maintaining its reading frame. Residues U₃₈ and U₃₉ in the anticodon stem–loop are frequently modified to pseudouridine (Ψ) by members of the widely conserved TruA/Pus3 family of pseudouridylases. We investigate here the cause of the temperature sensitivity of pus3Δ mutants of the yeast Saccharomyces cerevisiae and find that, although Ψ₃₈ or Ψ₃₉ is found on at least 19 characterized cytoplasmic tRNA species, the temperature sensitivity is primarily due to poor function of tRNA^Gln(UUG), which normally has Ψ₃₈. Further investigation reveals that at elevated temperatures there are substantially reduced levels of the s²U moiety of mcm⁵s²U₃₄ of tRNA^Gln(UUG) and the other two cytoplasmic species with mcm⁵s²U₃₄, that the reduced s²U levels occur in the parent strain BY4741 and in the widely used strain W303, and that reduced levels of the s²U moiety are detectable in BY4741 at temperatures as low as 33°C. Additional examination of the role of Ψ_38,39 provides evidence that Ψ₃₈ is important for function of tRNA^Gln(UUG) at permissive temperature, and indicates that Ψ₃₉ is important for the function of tRNA^Trp(CCA) in trm10Δ pus3Δ mutants and of tRNA^Leu(CAA) as a UAG nonsense suppressor. These results provide evidence for important roles of both Ψ₃₈ and Ψ₃₉ in specific tRNAs, and establish that modification of the wobble position is subject to change under relatively mild growth conditions.     

Construction,aminoacylation and 80 S ribosomal complex formation with a yeast initiator tRNA having an arginine CCU anticodon

The digestion of yeast initiator methionine tRNA with mung bean nuclease and U₂ ribonuclease yielded 5'- and 3'-fragments, respectively. These two fragments together represent the entire tRNA sequence except for A₃₅, the central nucleotide of the anticodon, and the CCA terminus. Using RNA ligase, a cytosine was added and the anticodon loop having a C₃₅ was reformed. Subsequent treatment of this product with CCA-transferase yielded a full-length methionine tRNA having an arginine CCU anticodon. This recombinant tRNA^Met (CCU) was charged with methionine by the yeast tRNA synthetase. Aminoacylation of the recombinant was however less extensive than in the case of native tRNA^Met (CAU). After aminoacylation the recombinant tRNA formed an 80 S ribosomal complex.     

Primary structure of three leucine transfer RNAs from bean chloroplast

Three tRNAs^Leu have been purified from bean chloroplasts and their nucleotide sequence determined. tRNA₁^Leu has 88 nucleotides and a U¹AA anticodon, tRNA₂^Leu has 85 nucleotides and a CmAA anticodon, and tRNA₃^Leu has 83 nucleotides and a UAm⁷G anticodon.     

Solution structure of psi32-modified anticodon stem-loop of Escherichia coli tRNAPhe

Nucleoside base modifications can alter the structures and dynamics of RNA molecules and are important in tRNAs for maintaining translational fidelity and efficiency. The unmodified anticodon stem–loop from Escherichia coli tRNA^Phe forms a trinucleotide loop in solution, but Mg²⁺ and dimethylallyl modification of A₃₇ N6 destabilize the loop-proximal base pairs and increase the mobility of the loop nucleotides. The anticodon arm has three additional modifications, ψ₃₂, ψ₃₉, and A₃₇ C2-thiomethyl. We have used NMR spectroscopy to investigate the structural and dynamical effects of ψ₃₂ on the anticodon stem-loop from E.coli tRNA^Phe. The ψ₃₂ modification does not significantly alter the structure of the anticodon stem–loop relative to the unmodified parent molecule. The stem of the RNA molecule includes base pairs ψ₃₂-A₃₈ and U₃₃–A₃₇ and the base of ψ₃₂ stacks between U₃₃ and A₃₁. The glycosidic bond of ψ₃₂ is in the anti configuration and is paired with A₃₈ in a Watson–Crick geometry, unlike residue 32 in most crystal structures of tRNA. The ψ₃₂ modification increases the melting temperature of the stem by ~3.5°C, although the ψ₃₂ and U₃₃ imino resonances are exchange broadened. The results suggest that ψ₃₂ functions to preserve the stem integrity in the presence of additional loop modifications or after reorganization of the loop into a translationally functional conformation.     

An unusual tRNAThr derived from tRNAHis reassigns in yeast mitochondria the CUN codons to threonine

The standard genetic code is used by most living organisms, yet deviations have been observed in many genomes, suggesting that the genetic code has been evolving. In certain yeast mitochondria, CUN codons are reassigned from leucine to threonine, which requires an unusual tRNA^Thr with an enlarged 8-nt anticodon loop (). To trace its evolutionary origin we performed a comprehensive phylogenetic analysis which revealed that evolved from yeast mitochondrial tRNA^His. To understand this tRNA identity change, we performed mutational and biochemical experiments. We show that Saccharomyces cerevisiae mitochondrial threonyl-tRNA synthetase (MST1) could attach threonine to both and the regular , but not to the wild-type tRNA^His. A loss of the first nucleotide (G₋₁) in tRNA^His converts it to a substrate for MST1 with a K_m value (0.7 μM) comparable to that of (0.3 μM), and addition of G₋₁ to allows efficient histidylation by histidyl-tRNA synthetase. We also show that MST1 from Candida albicans, a yeast in which CUN codons remain assigned to leucine, could not threonylate , suggesting that MST1 has coevolved with . Our work provides the first clear example of a recent recoding event caused by alloacceptor tRNA gene recruitment.     

Crystal structure analysis reveals functional flexibility in the selenocysteine-specific tRNA from mouse

Background

Selenocysteine tRNAs (tRNA^Sec) exhibit a number of unique identity elements that are recognized specifically by proteins of the selenocysteine biosynthetic pathways and decoding machineries. Presently, these identity elements and the mechanisms by which they are interpreted by tRNA^Sec-interacting factors are incompletely understood.

Methodology/Principal Findings

We applied rational mutagenesis to obtain well diffracting crystals of murine tRNA^Sec. tRNA^Sec lacking the single-stranded 3′-acceptor end (^ΔGCCARNA^Sec) yielded a crystal structure at 2.0 Å resolution. The global structure of ^ΔGCCARNA^Sec resembles the structure of human tRNA^Sec determined at 3.1 Å resolution. Structural comparisons revealed flexible regions in tRNA^Sec used for induced fit binding to selenophosphate synthetase. Water molecules located in the present structure were involved in the stabilization of two alternative conformations of the anticodon stem-loop. Modeling of a 2′-O-methylated ribose at position U34 of the anticodon loop as found in a sub-population of tRNA^Sec in vivo showed how this modification favors an anticodon loop conformation that is functional during decoding on the ribosome. Soaking of crystals in Mn²⁺-containing buffer revealed eight potential divalent metal ion binding sites but the located metal ions did not significantly stabilize specific structural features of tRNA^Sec.

Conclusions/Significance

We provide the most highly resolved structure of a tRNA^Sec molecule to date and assessed the influence of water molecules and metal ions on the molecule''s conformation and dynamics. Our results suggest how conformational changes of tRNA^Sec support its interaction with proteins.     

The crystal structure of yeast mitochondrial ThrRS in complex with the canonical threonine tRNA

In mitochondria of Saccharomyces cerevisiae, a single aminoacyl-tRNA synthetase (aaRS), MST1, aminoacylates two isoacceptor tRNAs, tRNA₁^Thr and tRNA₂^Thr, that harbor anticodon loops of different size and sequence. As a result of this promiscuity, reassignment of the CUN codon box from leucine to threonine is facilitated. However, the mechanism by which a single aaRS binds distinct anticodon loops with high specificity is not well understood. Herein, we present the crystal structure of MST1 in complex with the canonical tRNA₂^Thr and non-hydrolyzable analog of threonyl adenylate. Our structure reveals that the dimeric arrangement of MST1 is essential for binding the 5′-phosphate, the second base pair of the acceptor stem, the first two base pairs of the anticodon stem and the first nucleotide of the variable arm. Further, in contrast to the bacterial ortholog that ‘reads’ the entire anticodon sequence, MST1 recognizes bases in the second and third position and the nucleotide upstream of the anticodon sequence. We speculate that a flexible loop linking strands β4 and β5 may be allosteric regulator that establishes cross-subunit communication between the aminoacylation and tRNA-binding sites. We also propose that structural features of the anticodon-binding domain in MST1 permit binding of the enlarged anticodon loop of tRNA₁^Thr.     

Investigation of in vivo diferric tyrosyl radical formation in Saccharomyces cerevisiae Rnr2 protein: requirement of Rnr4 and contribution of Grx3/4 AND Dre2 proteins

The β₂ subunit of class Ia ribonucleotide reductase (RNR) contains a diferric tyrosyl radical cofactor (Fe₂^III-Tyr^•) that is essential for nucleotide reduction. The β₂ subunit of Saccharomyces cerevisiae is a heterodimer of Rnr2 (β) and Rnr4 (β′). Although only β is capable of iron binding and Tyr^• formation, cells lacking β′ are either dead or exhibit extremely low Tyr^• levels and RNR activity depending on genetic backgrounds. Here, we present evidence supporting the model that β′ is required for iron loading and Tyr^• formation in β in vivo via a pathway that is likely dependent on the cytosolic monothiol glutaredoxins Grx3/Grx4 and the Fe-S cluster protein Dre2. rnr4 mutants are defective in iron loading into nascent β and are hypersensitive to iron depletion and the Tyr^•-reducing agent hydroxyurea. Transient induction of β′ in a GalRNR4 strain leads to a concomitant increase in iron loading and Tyr^• levels in β. Tyr^• can also be rapidly generated using endogenous iron when permeabilized Δrnr4 spheroplasts are supplemented with recombinant β′ and is inhibited by adding an iron chelator prior to, but not after, β′ supplementation. The growth defects of rnr4 mutants are enhanced by deficiencies in grx3/grx4 and dre2. Moreover, depletion of Dre2 in GalDRE2 cells leads to a decrease in both Tyr^• levels and ββ′ activity. This result, in combination with previous findings that a low level of Grx3/4 impairs RNR function, strongly suggests that Grx3/4 and Dre2 serve in the assembly of the deferric Tyr^• cofactor in RNR.     

The molecular basis for the differential translation of TMV RNA in tobacco protoplasts and wheat germ extracts

Translation of tobacco mosaic virus (TMV) RNA in tobacco protoplasts yields the 17.5-K coat protein, a 126-K protein and a 183-K protein which is generated by an efficient readthrough over the UAG termination codon at the end of the 126-K cistron. In wheat germ extracts, however, only the 5'-proximal 126-K cistron is translated whereas the 183-K readthrough protein is not synthesized. Purification and sequence analysis of the endogenous tyrosine tRNAs revealed that the uninfected tobacco plant contains two tRNAs^Tyr, both with GΨA anticodons which stimulate the UAG readthrough in vitro and presumably in vivo. In contrast, ˜85% of the tRNA^Tyr from wheat germ contains a QΨA anticodon and ˜15% has a GΨA anticodon. Otherwise the sequences of tRNAs^Tyr from wheat germ and tobacco are identical. UAG readthrough and hence synthesis of the 183-K protein is only stimulated by tRNA^Tyr_GΨA and not at all by tRNA^Tyr_QΨA. The tRNAs^Tyr from wheat leaves were also sequenced. This revealed that adult wheat contains tRNA^Tyr_GΨA only. This is very much in contrast to the situation in animals, where Q-containing tRNAs are characteristic for adult tissues whereas Q deficiency is typical for the neoplastic and embryonic state.     

A critical role of water in the specific cleavage of the anticodon loop of some eukaryotic methionine initiator tRNAs

We have noticed that during a long storage and handling, the plant methionine initiator tRNA is spontaneously hydrolyzed within the anticodon loop at the C34-A35 phosphodiester bond. A literature search indicated that there is also the case for human initiator tRNA^Met but not for yeast tRNA^Met _i or E. coli tRNA^Met _f. All these tRNAs have an identical nucleotide sequence of the anticodon stems and loops with only one difference at position 33 within the loop. It means that cytosine 33 (C33) makes the anticodon loop of plant and human tRNA^Met _i susceptible to the specific cleavage reaction. Using crystallographic data of tRNA^Met _f of E. coli with U33, we modeled the anticodon loop of this tRNA with C33. We found that C33 within the anticodon loop creates a pocket that can accomodate a hydrogen bonded water molecule that acts as a general base and catalyzes a hydrolysis of C-A bond. We conclude that a single nucleotide change in the primary structure of tRNA^Met _i made changes in hydration pattern and readjustment in hydrogen bonding which lead to a cleavage of the phosphodiester bond.     

Sequence studies on human placenta tRNAVal : comparison with the mouse myeloma tRNAVal

The major species of valine specific tRNA was isolated from human placenta, degraded to oligonucleotides, and shown to have the nucleotide sequence pG-U-U-U-C-C-G-U-A-G-U-G-U?-A-G-D-G-G-D-D-A-U-C-A-C-m²G-U?-U-C-G-C-C-U-(I or C)-A-C-A-C-G-C-G-A-A-A-G-m⁷G-D-m⁵C-m⁵C-C-C-G-G-U-U?-C-G-m¹A-A-A-C-C-G-G-G-C-G-G-A-A-A-C-A-C-C-A_OH. This human placental tRNA^Val differs from the major species of mouse myeloma tRNA^Val only in that it contains either I or C in the wobble position of the anticodon, and totally lacks 2′-O-methylcytosine and 5-methylcytosine in the anticodon loop.     

Conformational Change of the L-Shaped tRNAPhe Molecule

Abstract

Fluorophore of proflavine was introduced onto the 3′-terminal ribose moiety of yeast tRNA^Phe. The distance between the fluorophore and the fluorescent Y base in the anticodon of yeast tRNA^Phe was measured by a singlet-singlet energy transfer. Conformational changes of tRNA^Phe with binding of tRNA^Glu ₂, which has the anticodon UUC complementary to the anticodon GAA of tRNA^Phe, were investigated. The distance obtained at the ionic strength of 100 mM K⁺ and 10 mM Mg²⁺ is very close to the distance from x-ray diffraction, while the distance obtained in the presence of tRNA^Glu ₂ is significantly smaller. Further, using a fluorescent probe of 4-bromomethl-7-methoxycoumarin introduced onto pseudouridine residue Ψ₅₅ in the TΨC loop of tRNA^Phe, Stern-Volmer quenching experiments for the probe with or without added tRNA^Glu ₂were carried out. The results showed greater access of the probe to the quencher with added tRNA^Glu ₂. These results suggest that both arms of the L-shaped tRNA structure tend to bend inside with binding of tRNA^Glu ₂ and some structural collapse occurs at the corner of the L-shaped structure.     

The genotype of early-transmitting HIV gp120s promotes α (4) β(7)-reactivity, revealing α (4) β(7) +/CD4+ T cells as key targets in mucosal transmission

Mucosal transmission of HIV is inefficient. The virus must breach physical barriers before it infects mucosal CD4⁺ T cells. Low-level viral replication occurs initially in mucosal CD4⁺ T cells, but within days high-level replication occurs in Peyer''s patches, the gut lamina propria and mesenteric lymph nodes. Understanding the early events in HIV transmission may provide valuable information relevant to the development of an HIV vaccine. The viral quasispecies in a donor contracts through a genetic bottleneck in the recipient, such that, in low-risk settings, infection is frequently established by a single founder virus. Early-transmitting viruses in subtypes A and C mucosal transmission tend to encode gp120s with reduced numbers of N-linked glycosylation sites at specific positions throughout the V1-V4 domains, relative to typical chronically replicating isolates in the donor quasispecies. The transmission advantage gained by the absence of these N-linked glycosylation sites is unknown. Using primary α₄β₇ ⁺/CD4⁺ T cells and a flow-cytometry based steady-state binding assay we show that the removal of transmission-associated N-linked glycosylation sites results in large increases in the specific reactivity of gp120 for integrin- α₄β₇. High-affinity for integrin α₄β₇, although not found in many gp120s, was observed in early-transmitting gp120s that we analyzed. Increased α₄β₇ affinity is mediated by sequences encoded in gp120 V1/V2. α₄β₇-reactivity was also influenced by N-linked glycosylation sites located in C3/V4. These results suggest that the genetic bottleneck that occurs after transmission may frequently involve a relative requirement for the productive infection of α₄β₇ ⁺/CD4⁺ T cells. Early-transmitting gp120s were further distinguished by their dependence on avidity-effects to interact with CD4, suggesting that these gp120s bear unusual structural features not present in many well-characterized gp120s derived from chronically replicating viruses. Understanding the structural features that characterize early-transmitting gp120s may aid in the design of an effective gp120-based subunit vaccine.     

Processing of plant mitochondrial tRNAGly and tRNASer(GCU) is independent of RNA editing

The genes encoding pea and potato mitochondrial tRNA^Gly and pea mitochondrial tRNA^Ser(GCU) were analyzed with particular respect to their expression. Secondary-structure models deduced from the identical potato and pea tRNA^Gly gene sequences revealed A₇:C₆₆ mismatches in the seventh base pair at the base of the acceptor stems of both tRNAs. Sequence analyses of tRNA^Gly cDNA clones showed that these mispairings are not corrected by C₆₆ to U₆₆ conversions, as observed in plant mitochondrial tRNA^Phe. Likewise, a U₆:C₆₇ mismatch identified in the acceptor stem of the pea tRNA^Ser(GCU) is not altered by RNA editing to a mismatched U:U pair, which is created by RNA editing in Oenothera mitochondrial tRNA^Cys. In vitro processing reactions with the respective tRNA^Gly and tRNA^Ser(GCU) precursors show that such conversions are not necessary for 5′ and 3′ end maturation of these tRNAs. These results demonstrate that not all C:A (A:C) or U:C (C:U) mismatches in double-stranded regions of tRNAs are altered by RNA editing. An RNA editing event in plant mitochondrial tRNAs is thus not generally indicated by the presence of a mismatch but may depend on additional parameters.     

Fidelity of tRNA 5'-maturation: a possible basis for the functional dependence of archaeal and eukaryal RNase P on multiple protein cofactors

RNase P, which catalyzes tRNA 5′-maturation, typically comprises a catalytic RNase P RNA (RPR) and a varying number of RNase P proteins (RPPs): 1 in bacteria, at least 4 in archaea and 9 in eukarya. The four archaeal RPPs have eukaryotic homologs and function as heterodimers (POP5•RPP30 and RPP21•RPP29). By studying the archaeal Methanocaldococcus jannaschii RPR''s cis cleavage of precursor tRNA^Gln (pre-tRNA^Gln), which lacks certain consensus structures/sequences needed for substrate recognition, we demonstrate that RPP21•RPP29 and POP5•RPP30 can rescue the RPR''s mis-cleavage tendency independently by 4-fold and together by 25-fold, suggesting that they operate by distinct mechanisms. This synergistic and preferential shift toward correct cleavage results from the ability of archaeal RPPs to selectively increase the RPR''s apparent rate of correct cleavage by 11 140-fold, compared to only 480-fold for mis-cleavage. Moreover, POP5•RPP30, like the bacterial RPP, helps normalize the RPR''s rates of cleavage of non-consensus and consensus pre-tRNAs. We also show that archaeal and eukaryal RNase P, compared to their bacterial relatives, exhibit higher fidelity of 5′-maturation of pre-tRNA^Gln and some of its mutant derivatives. Our results suggest that protein-rich RNase P variants might have evolved to support flexibility in substrate recognition while catalyzing efficient, high-fidelity 5′-processing.     

Translation of the UGA triplet in vitro by tryptophan transfer RNA's

Tryptophan transfer RNA from the UGA-suppressing strain of Escherichia coli CAJ64 was purified and assayed for suppressor activity in vitro in two ways: by translation of the bacteriophage T4 lysozyme messenger RNA bearing a UGA mutation, and by translation of poly(U-G-A). Purified tRNA^Trp, and no other fraction, stimulates lysozyme synthesis 30-fold above the level seen when comparable amounts of tryptophan tRNA from the non-suppressing strain, CA244, were added; it also translates poly(U-G-A) as polytryptophan more efficiently than the su⁻ tRNA. Tryptophan tRNA from the non-suppressing strain is active in the assays but far less so than CAJ64 tRNA^Trp, and this is consistent with the leakiness of su⁻ strains. Since the nucleotide sequences of these tryptophan tRNA's are known (Hirsh, 1971), it is concluded that tRNA with a CCA anticodon recognizes the UGA triplet and this recognition is improved by a nucleotide change elsewhere in the molecule.     

A factor promoting the ejection of deacylated initiator transfer RNA from ribosomes

Experiments in bacterial cell-free extracts show that the deacylated initiator transfer RNA can be specifically discharged from its complex with the ribosome and messenger by addition of GTF and a factor derived from ribosomal salt-washings. This release-factor differs from that for the non-initiating tRNA's in its capability to dissociate the tRNA_f^Met-30 s ribosome-ApUpG complex^‡, in its inability to be inhibited by fusidic acid, and in its Chromatographic mobility on an ion-exchange column.     

Conformation effects of base modification on the anticodon stem-loop of Bacillus subtilis tRNA(Tyr)

tRNA molecules contain 93 chemically unique nucleotide base modifications that expand the chemical and biophysical diversity of RNA and contribute to the overall fitness of the cell. Nucleotide modifications of tRNA confer fidelity and efficiency to translation and are important in tRNA-dependent RNA-mediated regulatory processes. The three-dimensional structure of the anticodon is crucial to tRNA-mRNA specificity, and the diverse modifications of nucleotide bases in the anticodon region modulate this specificity. We have determined the solution structures and thermodynamic properties of Bacillus subtilis tRNA^Tyr anticodon arms containing the natural base modifications N⁶-dimethylallyl adenine (i⁶A₃₇) and pseudouridine (ψ₃₉). UV melting and differential scanning calorimetry indicate that the modifications stabilize the stem and may enhance base stacking in the loop. The i⁶A₃₇ modification disrupts the hydrogen bond network of the unmodified anticodon loop including a C₃₂-A₃₈⁺ base pair and an A₃₇-U₃₃ base-base interaction. Although the i⁶A₃₇ modification increases the dynamic nature of the loop nucleotides, metal ion coordination reestablishes conformational homogeneity. Interestingly, the i⁶A₃₇ modification and Mg²⁺ are sufficient to promote the U-turn fold of the anticodon loop of Escherichia coli tRNA^Phe, but these elements do not result in this signature feature of the anticodon loop in tRNA^Tyr.