Removal of Y-37 from tRNA phe yeast alters oligomer binding to two loops

The association constants of complementary oligomers were used to monitor changes in the structure of tRNA_yeast^phe as a consequence of excision of a single base Y in the anticodon loop and of clipping the molecule at the point of excision. Significant changes were found not only in the binding constants of oligomers complementary to the anticodon loop but also in the K of an oligomer complementary to the dihydro U loop. The results suggest that either a single base change in a tRNA may alter structure elsewhere in the molecule or that the acid treatment necessary to remove Y irreversibly alters the structure of tRNA_yeast^phe.     

Study of the phosphorescent bases of yeast phenylalanine transfer RNA with the aid of optical detection of magnetic resonance

The phosphorescence of brewers' yeast phenylalanine transfer RNA has been investigated at 77 °K and at 1.2 °K in pumped liquid helium. Although the phosphorescence at 77 °K originates almost completely from the Y base in the anticodon loop, independent of excitation wavelength, the phosphorescence originates from normal bases with 270 nm excitation at temperatures in the helium range. The low-temperature phosphorescence is assigned to the triplet state of adenosine by optical detection of magnetic resonance measurements. The adenosine phosphorescence at 1.2 °K is quenched by the binding of the codon poly(U), as well as by the removal of Mg²⁺. The former result indicates that the adenosine phosphorescence originates from the anticodon, -Gm-A-A-, while the second shows that a conformational change introduced by removing Mg²⁺ (possibly involving unstacking of the anticodon) prevents energy trapping in the anticodon triplet state. The lack of triplet energy transfer from anticodon to Y indicates that Y cannot be stacked with the anticodon in the conformation that is stable at helium temperature. The adenosine phosphorescence of transfer RNA^Phe is nearly completely quenched at 77 °K, at least partially due to energy transfer to Y. We think that the thermally activated energy transfer is associated with some mobility of the Y base at 77 °K. Our observations are in contrast with previous results on bakers' yeast tRNA^Phe where there is apparently little, if any, energy transfer to Y from the normal nucleotides at 80 °K with 265 nm excitation. Optically detected magnetic resonance measurements on the triplet state of Y base in various environments indicate that removal of Mg²⁺ causes a shift of the Y base in tRNA^Phe to a more solvent-exposed position, whereas the binding of poly(U) has little effect on the environment of Y.     

The structure of the anticodon loop of tRNAPhe from yeast as deduced from spectroscopic studies on oligonucleotides

The hexanucleotide Gm-A-A-Y-A-ψp excised from the anticodon loop of yeast tRNA^Phe and its constituent oligonucleotides have been studied by ultraviolet absorption spectroscopy, static fluorescence, and circular dichroism. Gm-Ap has a melting point of 45°C and a high melting enthalpy when compared with G-Ap; hence 2′-O-methylation seems to stabilize stacking interactions. The nucleobase Y adjacent to the 3′-side of the anticodon triplet interacts stronger with its 3′-neighboring A than with its 5′-neighboring A. It is concluded that the base Y disconnects the stack of the anticodon itself from the stack of the anticodon stem, thereby setting a reading frame for the mRNA in the course of protein biosynthesis. From the opposite signs of the short-wavelength Cotton effects in the spectra of Gm-A-A-Y-Ap and Gm-A-A-Y, it is concluded that Y after removal of its 3′ neighbor undergoes a dramatic change in its conformation. The fluorescence of the nucleobase Y upon addition of Mg²⁺ is enhanced in oligonucleotides longer than two. An identical enhancement is observed for tRNA^Phe, indicating that this Mg²⁺ effect is a property of an oligonucleotide segment and does not reflect conformational changes of the whole tRNA. The data presented here reveal that the basic structural features of the anticodon loop are already present in the hexanucleotide Gm-A-A-Y-A-ψp and are not determined by the overall structure of tRNA.     

Targeting different RNA motifs by beta carboline alkaloid,harmalol: a comparative photophysical,calorimetric, and molecular docking approach

RNA has attracted recent attention for its key role in gene expression and targeting by small molecules for therapeutic intervention. This work focuses towards understanding interaction of harmalol, a DNA intercalator, with RNAs of different motifs viz. single-stranded A-form poly(A), double-stranded A-form of poly(C)·poly(G), and clover leaf tRNA^phe by different spectroscopic, calorimetric, and molecular modeling techniques. Results of this study converge to suggest that (i) binding constant varied in the order poly(C)·poly(G)?>?tRNA^phe > poly(A), (ii) non-cooperative binding of harmalol to poly(C)·poly(G) and poly(A) and cooperative binding with tRNA^phe, (iii) significant structural changes of poly(C)·poly(G) and tRNA^phe with concomitant induction of optical activity in the bound achiral alkaloid molecules, while with poly(A) no induced Circular dichroism (CD) perturbation was observed, (iv) the binding was predominantly exothermic, enthalpy-driven, entropy-favored with poly(C)·poly(G), while it was entropy driven with tRNA^phe and poly(A), (v) a hydrophobic contribution and comparatively large role of non polyelectrolytic forces to Gibbs energy changes with poly(C)·poly(G) and tRNA^phe and (vi) intercalated state of harmalol inside poly(C)·poly(G) structure as revealed from molecular docking was supported by the viscometric and ferrocyanide quenching data. All these findings unequivocally pointed out that harmalol prefers binding with poly(C)·poly(G), compared to tRNA^phe and poly(A); this results serve as data for the development of RNA-based antiviral drugs.     

Activation of several tRNAs of Escherichia coli by the phenoxyacetyl derivative of N-hydroxysuccinimide

Escherichia coli 15T^? treated with chloramphenicol produces tRNA^phe which is deficient in minor nucleosides. Undermodified tRNA^phe chromatographs as two new peaks from a benzoylated diethylaminoethyl-cellulose column. Chloramphenicol tRNA^phe was purified by phenoxyacetylation of phenylalanyl-tRNA and subsequent chromatography on benzoylated diethylaminoethyl-cellulose. Purified tRNA^phe had an altered Chromatographie profile as a result of the purification procedure. Phenoxyacetylation of an unpurified tRNA preparation, which was either charged with phenylalanine or kept discharged, resulted in a permanent alteration of tRNA^phe which was similar to the alteration of the purified tRNA^phe. The altered tRNAs eluted with higher salt or ethanol concentrations from benzoylated diethylaminoethyl-cellulose. The alteration was also shown for tRNA^phe of phenoxyacetylated tRNA from late log phase E. coli 15T?. tRNA^glu and tRNA^Leu were not changed, but both tRNA^Arg and tRNA^Ile were altered. tRNA₂^Val and tRNA^Met shifted in the elution profile; tRNA₁^Val and tRNA_f^Met were not affected.Comparison of the primary structures of the alterable and nonalterable tRNA's revealed that all alterable tRNA's have the undefined nucleoside X in the extra loop. Phenoxyacetylation of nucleoside X probably was the cause of the altered profiles.tRNA^phe from E. coli 15T^? treated with chloramphenicol was less reactive towards phenoxyacetylation than normal tRNA, possibly because of a different conformation of the modification-deficient molecule relative to the normal tRNA^phe. tRNA^phe from E. coli 15T^?, starved for cysteine and methionine and treated with chloram-phenicol, is more deficient in minor nucleosides and showed even less reactivity.Acceptor capacities of the altered tRNA species were not changed significantly; only the acceptor capacity for tRNA^Ile decreased approximately 25%. The recognition site for the aminoacyl-tRNA synthetases probably is not affected.     

The anticodon-anticodon complex

Gel electrophoresis has been used to measure the binding between two tRNAs with complementary anticodons, tRNA^Val (Escherichia coli) (anticodon X,A,C) and tRNA^Tyr (E. coli) (anticodon Q,U,A). The association constant K at 0 °C was found to be 4 × 10⁵ m^?1 which is about three orders of magnitude greater than the association constant for tRNA^Tyr (E. coli) binding its trinucleotide codon UAC. The temperature dependence of K suggests that this results from the rigidity of the anticodon loop. tRNA^Tyr (E. coli) binds an order of magnitude more weakly to tRNA^Val (yeast) than to tRNA^Val (E. coli), presumably because it contains the wobble base pair A · I. The relationship between the anticodon-anticodon complex and codon recognition is discussed.     

The structure of yeast tRNAAsp. A model for tRNA interacting with messenger RNA

Abstract

The anticodon of yeast tRNA^Asp, GUC, presents the peculiarity to be self-complementary, with a slight mismatch at the uridine position. In the orthorhombic crystal lattice, tRNA^Asp molecules are associated by anticodon-anticodon interactions through a two-fold symmetry axis. The anticodon triplets of symmetrically related molecules are base paired and stacked in a normal helical conformation. A stacking interaction between the anticodon loops of two two-fold related tRNA molecules also exists in the orthorhombic form of yeast tRNA^Phe. In that case however the GAA anticodon cannot be base paired. Two characteristic differences can be correlated with the anticodon-anticodon association: the distribution of temperature factors as determined from the X-ray crystallographic refinements and the interaction between T and D loops. In tRNA^Asp T and D loops present higher temperature factors than the anticodon loop, in marked contrast to the situation in tRNA^Phe. This variation is a consequence of the anticodon-anticodon base pairing which rigidities the anticodon loop and stem. A transfer of flexibility to the corner of the tRNA molecule disrupts the G19-C56 tertiary interactions. Chemical mapping of the N3 position of cytosine 56 and analysis of self-splitting patterns of tRNA^Asp substantiate such a correlation.     

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.     

Crosslinking of tRNAs by the Carcinostatic Agent Dirhodium Tetraacetate

Abstract

A crystalline complex of yeast tRNA^phe and dirhodium tetraacetate (DRTA) was prepared and its X-ray structure determined. The bifunctional DRTA forms an intermolecular crosslink between the N(1) position of adenine A36 in the anticodon triplet and possibly a ribose hydroxyl group of residue A76 at the 3′ terminus of a symmetry related tRNA molecule. The rhodium complex apparently shows a preference for binding to the N(l) position of adenine in a single strand region of the tRNA molecule.     

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.     

300 MHz PMR studies on the conformation of the hexanucleotide, 2'OMeGpApApUpApPsi, from the anticodon loop of torula yeast tRNAphe

A complete temperature study of the proton resonances of the hexanucleotide, 2′-OMeGpApApYpApψ, from Torula yeast tRNA^phe has been carried out at 300 MHz. The data has been interpreted in terms of a base stacked oligomer in which the glycosyl conformation of the Y-nucleoside changes from syn to anti with temperature increase. An alternative structure for the Y-base is proposed to permit this conformational change.     

The distance between the anticodon loops of two tRNAs bound to the 70 S Escherichia coli ribosome.

Using singlet-singlet energy transfer, we have measured the distance between the anticodons of two transfer RNAs simultaneously bound to a messengerprogramed Escherichia coli 70 S ribosome. The fluorescent Y base adjacent to the anticodon of yeast tRNA_Y^Phe serves as a donor. A proflavine (Pf) chemically substituted for the Y base in tRNA_Pf^Phe serves as an acceptor. By exploiting the sequential binding properties of 70 S ribosomes for two deacylated tRNAs, we can fill the strong site with either tRNA_Y^Phe or tRNA_Pf^Phe and then the weak site with the other tRNA. In both cases donor quenching and sensitized emission of the acceptor are observed. Analysis of these results leads to an estimate for the Y-proflavine distance of 18 ± 2 Å. This distance is very short and suggests strongly that the two tRNAs are simultaneously in contact with adjacent codons of the message. Separate experiments show that binding of a tRNA to the weak site does not perturb the environment of the hypermodified base of a tRNA bound to the strong site. This supports the assignment of the strong site as the peptidyl site. It also indicates that binding of the second tRNA proceeds without a change in the anticodon structure of a pre-existing tRNA at the peptidyl site.     

The Effect of Modification of Nucleotide-37 on the Interaction of Aminoacyl-tRNA with the A Site of the 70S Ribosome

To estimate the effect of modified nucleotide 37, the interaction of two yeast aminoacyl-tRNAs (Phe-tRNA^Phe _+Y and Phe-tRNA^Phe _–Y) with the A site of complex [70S · poly(U) · deacylated tRNA^Phe in the P site] was assayed at 0–20°C. As comparisons with native Phe-tRNA^Phe _+Y showed, removal of the Y base decreased the association constant of Phe-tRNA^Phe _–Y and the complex by an order of magnitude at every temperature tested, and increased the enthalpy of their interaction by 23 kJ/mol. When the Y base was present in the anticodon loop of deacylated tRNA^Phe bound to the P site of the 70S ribosome, twice higher affinity for the A site was observed for Phe-tRNA^Phe _–Y but not for Phe-tRNA^Phe _+Y. Thus, the modified nucleotide 3" of the Phe-tRNA^Phe anticodon stabilized the codon–anticodon interaction both in the A and P sites of the 70S ribosome.     

Function of Y in codon-anticodon interaction of tRNA Phe

Molar association constants of binding oligonucleotides to the anticodon loops of (yeast) tRNA^Phe, (yeast) tRNA_HCl^Phe and (E. coli) tRNA_F^Met have been determined by equilibrium dialysis. From the temperature dependence of the molar association constants, ΔF, ΔH and ΔS of oligomer-anticodon loop interaction have been determined. The data indicate that the free energy change of codon-anticodon interaction is highly influenced by the presence of a modified purine (tRNA^Phe), of an unmodified purine (tRNA_F^Met) or its absence (tRNA_HCl^Phe). Excision of the modified purine Y in the anticodon loop of tRNA^Phe results in a conformational change of the anticodon loop, which is discussed on the basis of the corresponding changes in ΔF, ΔH and ΔS.     

Binding of the 9-O-N-aryl/arylalkyl Amino Carbonyl Methyl Substituted Berberine Analogs to tRNAphe

Background

Three new analogs of berberine with aryl/arylalkyl amino carbonyl methyl substituent at the 9-position of the isoquinoline chromophore along with berberrubine were studied for their binding to tRNA^phe by wide variety of biophysical techniques like spectrophotometry, spectrofluorimetry, circular dichroism, thermal melting, viscosity and isothermal titration calorimetry.

Methodology/Principal Findings

Scatchard binding isotherms revealed that the cooperative binding mode of berberine was propagated in the analogs also. Thermal melting studies showed that all the 9-O-N-aryl/arylalkyl amino carbonyl methyl substituted berberine analogs stabilized the tRNA^phe more in comparison to berberine. Circular dichroism studies showed that these analogs perturbed the structure of tRNA^phe more in comparison to berberine. Ferrocyanide quenching studies and viscosity results proved the intercalative binding mode of these analogs into the helical organization of tRNA^phe. The binding was entropy driven for the analogs in sharp contrast to the enthalpy driven binding of berberine. The introduction of the aryl/arylalkyl amino carbonyl methyl substituent at the 9-position thus switched the enthalpy driven binding of berberine to entropy dominated binding. Salt and temperature dependent calorimetric studies established the involvement of multiple weak noncovalent interactions in the binding process.

Conclusions/Significance

The results showed that 9-O-N-aryl/arylalkyl amino carbonyl methyl substituted berberine analogs exhibited almost ten folds higher binding affinity to tRNA^phe compared to berberine whereas the binding of berberrubine was dramatically reduced by about twenty fold in comparison to berberine. The spacer length of the substitution at the 9-position of the isoquinoline chromophore appears to be critical in modulating the binding affinities towards tRNA^phe.     

Interaction of polyamines with fragments and whole molecules of yeast phenylalanine-specific transfer RNA

Binding of the polyamines spermidine (∼-+3) and spermine (∼-+4) to yeast tRNA^phe has been investigated by equilibrium dialysis under the same conditions used to study Mn²⁺-tRNA^phe interactions (Schreier & Schimmel, 1974). The polyamines bind to tRNA^phe in a co-operative and a non-co-operative phase, which is analogous to the behavior found with Mn²⁺. In the co-operative phase, the empirical index of co-operativity is somewhat greater for the polyamines, however. Binding constants for both the co-operative and non-co-operative phases are similar for Mn²⁺ and spermidine, and are strongest for spermine. Estimates of the total number of ligand binding sites indicate that these numbers are inversely proportional to the charge on the ligand for all three ligands. The interaction of polyamines with four large fragments of tRNA^phe shows no evidence for co-operativity. These results, together with recent kinetic studies, collectively suggest that polyamine binding to the co-operative sites is associated with tertiary structure formation and that polyamine and divalent metal ion interactions with tRNA occur by phenomenologically similar mechanisms, in spite of their structural diversity.     

Acceptor stem and anticodon RNA hairpin helix interactions with glutamine tRNA synthetase

The class I glutamine (Gln) tRNA synthetase interacts with the anticodon and acceptor stem of glutamine tRNA. RNA hairpin helices were designed to probe acceptor stem and anticodon stem-loop contacts. A seven-base pair RNA microhelix derived from the acceptor stem of tRNA^Gln was aminoacylated by Gln tRNA synthetase. Variants of the glutamine acceptor stem microhelix implicated the discriminator base as a major identity element for glutaminylation of the RNA helix. A second RNA microhelix representing the anticodon stem-loop competitively inhibited tRNA^Gln charging. However, the anticodon stem-loop microhelix did not enhance aminoacylation of the acceptor stem microhelix. Thus, transduction of the anticodon identity signal may require covalent continuity of the tRNA chain to trigger efficient aminoacylation.     

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.     

Comparison of the structures of the native form of rat liver 5S rRNA and yeast tRNAphe: small-angle and wide-angle X-ray scattering study

The small-angle and wide-angle X-ray scattering of tRNA^phe (yeast) and ribosomal 5S RNA (rat liver) in solution have been analysed and compared. tRNA^phe in solution is folded into a compact L-shaped structure similar to its structure in crystals. The geometry of the secondary structure of the double helical regions is also equivalent to the A-form in the crystalline state. Despite differences between the molar mosses of 5S rRNA (40 000 g mol^?1) and tRNA^phe (25 000 g mol^?1), and the fact that the 5S rRNA molecule is more anisometric than the tRNA^phe molecule, there are many structural similarities. The geometrical parameters of the secondary structure of double helical regions in both RNA molecules are almost identical; the mean rise per base pair is about 0.253–0.28 nm and the mean turn angle is about 32.5–33.5. Identical cross-sectional radii of gyration, R_sq,1 ≈ 1.16 nm and R_sq,2 = 0.92 nm, identical molar mass per unit length, , and a mean thickness of the molecules D ≈ 1.65 nm suggest a similar, nearly coplanar organization of isolated, double helical arms. Furthermore, there are compact regions in the central parts of both molecules, which are the sites of tertiary interactions in the tRNA^phe molecule and are a potential site of tertiary interactions in the SS rRNA molecule for stabilization of the complicated L-shape of the two molecules. Both molecules have a pseudo-twofold axis,w hich may play a role in recognition for binding of specific proteins.     

Mitochondrial Genome of <Emphasis Type="Italic">Ciona savignyi</Emphasis> (Urochordata,Ascidiacea, Enterogona): Comparison of Gene Arrangement and tRNA Genes with <Emphasis Type="Italic">Halocynthia roretzi</Emphasis> Mitochondrial Genome

The complete nucleotide sequence of the urochordate Ciona savignyi (Ascidiacea, Enterogona) mitochondrial (mt) genome (14,737 bp) was determined. The Ciona mt genome does not encode a gene for ATP synthetase subunit 8 but encodes an additional tRNA^Gly gene (anticodon UCU), as is the case in another urochordate, Halocynthia roretzi (Ascidiacea, Pleurogona), mt genome. In addition, the Ciona mt genome encodes two tRNA^Met genes; anticodon CAT and anticodon TAT. The tRNA^Cys gene is thought to lack base pairs at the D-stem. Thus, the Ciona mt genome encodes 12 protein, 2 rRNA, and 24 tRNA genes. The gene arrangement of the Ciona mt genome differs greatly from those of any other metazoan mt genomes reported to date. Only three gene boundaries are shared between the Halocynthia and the Ciona mt genomes. Molecular phylogenetic analyses based on amino acid sequences of mt protein genes failed to demonstrate the monophyly of the chordates.