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.     

Sequence analysis of three tRNAPhe nuclear genes and a mutated gene,and one gene for tRNAAla from Arabidopsis thaliana

Three genes and one mutant gene for tRNA^Phe (GAA) and one gene for tRNA^Ala (UGC) were isolated from a whole-cell DNA library of Arabidopsis thaliana. All three tRNA^Phe genes are identical in their nucleotide sequence, but differ in their 5 and 3 flanking regions. The mutant tRNA^Phe (GAA) gene differs from the other three genes by one nucleotide change from highly conserved G to C at the 57th nucleotide position. The primary structure of the first tRNA^Ala gene was also determined in this experiment.     

Transfer RNA Identity Change in Anticodon Variants of E. coli tRNAPhe in Vivo

The anticodon sequence is a major recognition element for most aminoacyl-tRNA synthetases. We investigated the in vivo effects of changing the anticodon on the aminoacylation specificity in the example of E. coli tRNA^Phe. Constructing different anticodon mutants of E. coli tRNA^Phe by site-directed mutagenesis, we isolated 22 anticodon mutant tRNA^Phe; the anticodons corresponded to 16 amino acids and an opal stop codon. To examine whether the mutant tRNAs had changed their amino acid acceptor specificity in vivo, we tested the viability of E. coli strains containing these tRNA^Phe genes in a medium which permitted tRNA induction. Fourteen mutant tRNA genes did not affect host viability. However, eight mutant tRNA genes were toxic to the host and prevented growth, presumably because the anticodon mutants led to translational errors. Many mutant tRNAs which did not affect host viability were not aminoacylated in vivo. Three mutant tRNAs containing anticodon sequences corresponding to lysine (UUU), methionine (CAU) and threonine (UGU) were charged with the amino acid corresponding to their anticodon, but not with phenylalanine. These three tRNAs and tRNA^Phe are located in the same cluster in a sequence similarity dendrogram of total E. coli tRNAs. The results support the idea that such tRNAs arising from in vivo evolution are derived by anticodon change from the same ancestor tRNA.     

Solvent accessibility study on tRNAPhe

In order to assess the solvent–solute association in the tRNA^Phe molecule, solvent accessibility calculations were carried out for its crystalline and completely extended states following the method of Lee and Richards. To do this, results from the calculations on model trinucleotide systems pApXpA with different bases at position X were used. In the folded form of the molecule, it was found that the oxygen atoms O(I) and O(II) of almost all the phosphate groups and the O(2′) atoms of the sugar rings situated throughout the backbone were highly exposed to the solvent. The amount of reduction found in the solvent accessibilities of the various atoms in going from the extended state to the folded state of the molecule indicates the kind of compactness of the tertiary structure in tRNA^Phe. The results give quantitative support to many characteristics of the tRNA molecule, such as loop sections, buried/exposed residues, hydrophobic interactions, etc., which were thought to be due to other factors.     

The conformation of the anticodon loop of yeast tRNAPhe in solution and on ribosomes.

The Y-base of yeast tRNA^Phe was replaced by the fluorophores 1-aminoanthracene or proflavine to yield derivatives which are active in all of the reactions of peptide elongation on reticulocyte ribosomes. The relatively long lifetime, higher quantum yield, and environmental sensitivity of 1-aminoanthracene make it a particulary useful adjunct to the Y-base in studying conformational changes in the anticodon region. The absorption and emission spectra of 1-aminoanthracene in tRNA in solutions in which it is active in peptide synthesis indicate that the probe is in a hydrophobic environment, apparently provided by stacking with the adjacent bases in the anticodon loop. The proflavine derivative, tRNA, was employed in iodide quenching, D₂O enhancement, and fluorescence depolarization experiments. The results indicate that the fluorophore in partially but not completely protected from the solvent. Anisotropy studies indicate that in solutions approximating those which support peptide synthesis on ribosomes, the probes have significant but restricted flexibility within the anticodon loop. Considered with nmr data and Y-base fluorescence from crystals of tRNA, the results indicate that the solution and crystal structures of tRNA^Phe are very similar. In turn, fluorescene from modified tRNA^Phe bound to ribosomes is similar to that observed in solution. It is of special significance for future experiments involving nonradiative energy transfer that these probles adjacent to the anticodon retain independent flexibility when bound to ribosomes with poly(U). The tRNA^Phe itself appears to be held rigidly on the ribosomes. It is concluded that within the limits dictated by the position and sensitivity of the probes used in this study, the mechanism of tRNA^Phe binding to ribosomes and the movement of tRNA and mRNA during the translocation steps of peptide synthesis can be interpreted in terms of the well-defined crystal structure of tRNA^Phe.     

Isolation and chromatographic behaviour of phenylalanine tRNA from barley embryos

Two fractions of phenylalanine tRNA (tRNA^Phe₁ and tRNA^Phe₂) were purified by BD-cellulose and RPC-5 chromatography of crude tRNA isolated from barley embryos. Successive RPC-5 rechromatography runs of tRNA^Phe₂ showed its conversion into more stable tRNA^Phe₁, suggesting that the two fractions have essentially the same primary structure. Both tRNA^Phe₁ and tRNA^Phe₂ had about the same acceptor activity, but tRNA^Phe₂ was aminoacylated much faster than tRNA^Phe₁. RPC-5 chromatography of crude aminoacylated tRNA showed higher contents of phe-tRNA^Phe₂ than of phe-tRNA^Phe₁ but the ratio of these two fractions estimated by relative fluorescence intensity was about 1. Fluorescence spectra of tRNA^Phe from barley embryos suggest that it contains Y base similar to Y_w from wheat tRNA^Phe.     

Variations during leaf development of the relative amounts of two bean (Phaseolus vulgaris) chloroplast tRNAsPhe which differ in their minor nucleotide content

Bean (Phaseolus vulgaris cv. Saxa) chloroplasts contain two tRNA^Phe species, namely tRNA^Phe1 and tRNA^Phe2. By sequence determination, we show that tRNA^Phe2 is identical to the previously sequenced tRNA^Phe1 except for two undermodified nucleotides. By reversed-phase chromatography analyses, we demonstrate that the relative amounts of these two chloroplast tRNAs^Phe vary during leaf development: in etiolated leaves the undermodified tRNA^Phe2 only represents 15% of total chloroplast tRNA^Phe, during development and greening it increases to reach 60% in 8-day-old leaves, and it then decreases to 9% in senescing leaves.     

Primary structure of Bombyx mori posterior silkgland tRNAPhe

tRNA^Phe was isolated from posterior silkgland from Bombyx mori and hydrolysed to mixtures of oligonucleotides. |³²P|5′ end labelling of the oligonucleotides and sequence study indicates that the major component of Bombyx mori tRNA^Phe is similar to mammalian tRNA^Phe, the minor component differing from the major one by one nucleotide only.     

N2-guanine specific transfer RNA methyltransferase II from rat liver

An enzyme was purified from rat liver and leukemic rat spleen which methylates guanosine residues in tRNA to N²-methylguanosine. By sequence analysis of bulk E. coli tRNA methylated with crude extracts it was shown that the enzyme is responsible for about 50% of total m²G formed invitro. The extent of methylation of a number of homogenous tRNA species was measured using the purified enzyme from both sources. Among tested E. coli tRNAs only tRNA^Arg, tRNA^Phe, and tRNA^Val yielded significantly more m²G than the bulk tRNA. The K_m for tRNA^Arg in the methylation reaction with enzymes from either tissue was 7.8 × 10⁻⁷ M as compared to the value 1 × 10⁻⁵ M obtained for the bulk tRNA. In a pancreatic RNase digest of bulk tRNA as well as of pure tRNA^Arg, tRNA^Phe, and tRNA^Val, A-m²G-Cp was found to be the only sequence methylated. Thus, the mammalian methyltransferase specifically recognizes the guanylate residue at position 10 from the 5′-end contained in a sequence (s⁴)U-A-G-Cp. Furthermore, there is no change between the enzyme from normal liver and leukemic spleen in the affinity for tRNA, the methylating capacity, and tRNA site and sequence recognition specificity.     

Thermodynamics of transfer ribonucleic acids: the effect of sodium on the thermal unfolding of yeast tRNAPhe.

The thermal unfolding of yeast phenylalanine-specific tRNA (tRNA^Phe) has been calorimetrically investigated at several salt concentrations in the absence of magnesium. Application of the deconvolution theory of macromolecular conformational transitions allows calculation of the thermodynamic parameters of unfolding. It is demonstrated that the unfolding of tRNA^Phe occurs in a sequential fashion and that four separate transitions or five macromolecular thermodynamic states exist in the temperature range 8–72°C under the experimental conditions of these studies (0.067–0.52M Na⁺). The enthalpy and entropy changes between states and the relative population of each state as a function of temperature and salt concentration have been obtained. Sodium stabilizes the low-temperature conformations of tRNA^Phe. The increase in the melting temperatures of each transition is shown to be linearly dependent on the logarithm of sodium concentration. These results allow calculation of the “phase” diagram for the transitions as a function of salt concentration.     

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.     

Effects of dilute HCl on yeast tRNAPhe and E. coli tRNAfMet1

HCl treatment of yeast tRNA^Phe under conditions generally used for excision of `Y' base results in structure and conformation changes as monitored by line widths in the PMR spectra at 220 MHz and by optical rotation. Like exposure of E. coli tRNA^fMet₁ causes similar changes in the PMR spectra and optical rotation although no residues are eliminated. Electrophoresis in polyacrylamide gels provides evidence for aggregation in HCl-treated tRNA^fMet₁. One must thus consider a general effect of HCl exposure as well as possible residue removal in assessing induced structural and conformation changes in tRNA.     

Biosynthesis of wyosine derivatives in tRNAPhe of Archaea: role of a remarkable bifunctional tRNAPhe:m1G/imG2 methyltransferase

The presence of tricyclic wyosine derivatives 3′-adjacent to anticodon is a hallmark of tRNA^Phe in eukaryotes and archaea. In yeast, formation of wybutosine (yW) results from five enzymes acting in a strict sequential order. In archaea, the intermediate compound imG-14 (4-demethylwyosine) is a target of three different enzymes, leading to the formation of distinct wyosine derivatives (yW-86, imG, and imG2). We focus here on a peculiar methyltransferase (aTrm5a) that catalyzes two distinct reactions: N¹-methylation of guanosine and C⁷-methylation of imG-14, whose function is to allow the production of isowyosine (imG2), an intermediate of the 7-methylwyosine (mimG) biosynthetic pathway. Based on the formation of mesomeric forms of imG-14, a rationale for such dual enzymatic activities is proposed. This bifunctional tRNA:m¹G/imG2 methyltransferase, acting on two chemically distinct guanosine derivatives located at the same position of tRNA^Phe, is unique to certain archaea and has no homologs in eukaryotes. This enzyme here referred to as Taw22, probably played an important role in the emergence of the multistep biosynthetic pathway of wyosine derivatives in archaea and eukaryotes.     

Nuclear magnetic resonance studies of codon-anticodon interaction in tRNAPhe. I. Effect of binding complementary tetra and pentanucleotides to the anticodon

The effect of codon-anticodon interaction on the structure of two tRNA^Phe species was investigated by means of nuclear magnetic resonance spectroscopy. To this end n.m.r.² spectra of yeast and Escherichia coli tRNA^Phe were recorded in the absence and the presence of the oligonucleotides U-U-C-A, U-U-C-G and U-U-C-A-G, which all contain the sequence UUC complementary to the anticodon sequence GAA. The spectra of the hydrogen-bonded protons, the methyl protons and the internucleotide phosphorous nuclei served to monitor the structure of the anticodon loop and of the tRNA in the tRNA-oligonucleotide complex. From the changes in the methyl proton spectra and in the phosphorous spectra it could be concluded that the oligonucleotides bind to the anticodon. Moreover it turned out that the binding constants obtained from these n.m.r. experiments were, within experimental error, equal to the values obtained with other techniques. Using the resonances of the protons hydrogen-bonded between the oligonucleotide and the anticodon loop the structure of the latter could be studied. In particular, binding of the pentanucleotide U-U-C-A-G, which is complementary to the five bases on the 5′ side of the anticodon loop, resulted in the resolution of four to five extra proton resonances indicating that four to five base-pairs are formed between the pentanucleotide and the anticodon loop. The formation of five base-pairs was confirmed by an independent fluorescence binding study. The resonance positions of the hydrogen-bonded protons indicate, that an RNA double helix is formed by the anticodon loop and U-U-C-A-G with the five base-pairs forming a continuous stack. This structure can be accomodated in the so-called 5′ stacked conformation of the anticodon loop, a structure that has been suggested earlier as an alternative to the familiar 3′ stacked conformation in the crystal structure models of yeast tRNA^Phe. It turned out that structural adjustments of the anticodon loop to the binding of the oligonucleotides are propagated into the anticodon stem. The relevance of these results with respect to the mechanism of protein synthesis is discussed.     

Theoretical Exploration of Netropsin Binding to tRNAPhe

Abstract

Theoretical exploration of the possible interaction of netropsin with tRNA^Phe indicates that binding should occur preferentially with the major groove of the TψC stem of the macromolecule, specifically with the bases G51, U52, G53 and phosphates 52, 53, 61 and 62. This agrees with the recent crystallographic result of Rubin and Sundaralingam. It is demonstrated that the difference with respect to netropsin binding with B-DNA, where it occurs specifically in the minor groove of AT sequences, is due to the differences in the distribution of the electrostatic molecular potential generated by these different types of DNA: this potential is sequence dependent in B-DNA (located in the minor groove of AT sequences and the major groove of GC sequences), while it is sequence independent and always located in the major groove in A-RNA. The result demonstrates the major role of electrostatics in determining the location of the binding site.     

Study of the Mechanism of Interaction of Oligonucleotides with the 3′-Terminal Region of tRNA<Superscript>Phe</Superscript> by Computer Modeling

Three-dimensional atomic models of complexes between yeast tRNA^Phe and 10- or 15-mer oligonucleotides complementary to the 3′-terminal tRNA sequence have been constructed using computer modeling. It has been found that rapidly formed primary complexes appear when an oligonucleotide binds to the coaxial acceptor and T stems of the tRNA^Phe along the major groove, which results in the formation of a triplex. Long stems allow the formation of a sufficiently strong complex with the oligonucleotide, which delivers its 3′-terminal nucleotides to the vicinity of the T loop adjoining the stem. These nucleotides destabilize the loop structure and initiate conformational rearrangements involving local tRNA^Phe destruction and formation of the final tRNA^Phe-oligonucleotide complementary complex. The primary complex formation and the following tRNA^Phe destruction constitute the “molecular wedge” mechanism. An effective antisence oligonucleotide should consist of three segments—(1) complex initiator, (2) primary complex stabilizer, and (3) loop destructor—and be complementary to the (free end)/loop-stem-loop tRNA structural element.