Sequence studies on tRNAPhe from placenta: comparison with known sequences of tRNAPhe from other normal mammalian tissues.

Phenylalanine-specific tRNA was isolated from human placenta and degraded to mixtures of oligonucleotides. Tritium sequence analysis of the digestion products indicates that the sequence of human placenta tRNA^Phe is identical to that of calf liver tRNA^Phe and differs only slightly from that of rabbit liver tRNA^Phe.     

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.     

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.     

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.     

Nucleoside Modifications Affect the Structure and Stability of the Anticodon of tRNALys,3

Abstract

NMR spectroscopy was used to determine the solution structures of RNA oligonucleotides comprising the anticodon domain of tRNA^Lys,3. The structural effects of the pseudouridine modification at position 39 were investigated and are well correlated with changes in thermodynamic parameters. The loop conformation differs from that seen in tRNA^Phe and provides an explanation of the critical role of modification in this tRNA.     

Location of a platinum binding site in the structure of yeast phenylalanine transfer RNA

Trans-dichlorodiammineplatinum (II) reacts with yeast phenylalanine transfer RNA to yield a major platinum binding site. The tightly bound platinum has been located on the oligonucleotide Gm-A-A-Y-A-ψp containing the anticodon by standard fingerprinting methods using ³²P-labelled tRNA^Phe. This site corresponds to a single major platinum site identified during an X-ray crystallographic analysis of yeast tRNA^Phe. The solution studies have given confidence to the assignment of part of the 3 Å electron density map to the anticodon region of the molecular structure of yeast tRNA^Phe.     

Invasion of Strongly Binding Oligonucleotides into tRNA Structure

Abstract

Interaction of yeast tRNA^Phe with oligodeoxyribonucleotides containing 5-methylcytosine, 2-aminoadenine, and 5-propynyl-2′-deoxyuridine was investigated. The modified oligonucleotides show increased binding capacity although the association rates are similar for the modified and natural oligonucleotides. The most pronounced increase in association constant (70 times) due to the incorporation of the strongly binding units was achieved in the case of oligonucleotide complementary to the sequence 65–76 of the tRNA^phe.     

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.     

Domains of phenylalanyl-tRNA synthetase from Thermus thermophilus required for aminoacylation

The contribution of entire domains or particular amino acid residues of the phenylalanyl-tRNA synthetase (FRS) from Thermus thermophilus to the interaction with tRNA^Phe was studied. Removal of domain 8 of the β subunit resulted in drastic reduction of the dissociation constant of the FRS·tRNA^Phe complex. Neither the removal of arginine 2 of the β subunit, which makes the only major contact between domains β1–5 and the tRNA, nor the replacement of the conserved proline 473 by glycine had an influence on the aminoacylation activity of the FRS. Thus, the body comprising domains 1–5 of the β subunit may not be essential for efficient aminoacylation of tRNA^Phe by the FRS and rather be involved in other functions.     

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.     

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.     

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.     

Calorimetric investigations of the helix-coil conversion of phenylalaninespecific transfer ribonucleic acid

The enthalpy of the helix-coil conversion of phenylalaninespecific transfer ribonucleic acid from brewer's yeast (tRNA^Phe_{brewer's yeast}) has been measured using both an LKB 10700-2 batch miciocalorimeter and an adiabatic differential scanning calorimeter. In the mixing calorimeter the conversion from coil to helix was induced by mixing a tRNA^Phe solution with a solution containing an excess of MgSO₄. We measured the enthalpy of this reaction stepwise in the temperature range from +9 to +60° C. For the enthalpy of folding of tRNA^Phe from coil to helix this method yielded the remarkably high value of ?310 $\text{kcal}\text{mole}$ of tRNA^Phe. With the differential scanning calorimeter in which the helix-coil conversion is simply induced by raising the temperature we found a value of +240 $\text{kcal}\text{mole}$ of tRNA^Phe at a Tm value of 76° C and a value of +200 $\text{kcal}\text{mole}$ of tRNA^Phe at a T_m value of 50° C. A comparison of the apparent van't Hoff enthalpies with the calorimetrically measured enthalpies shows, that the cooperativity of the system increases continually with rising melting temperatures - which are achieved by increasing Mg²⁺ concentrations - reaching a constant value at about 57° C. Above this temperature value the thermodynamic behaviour of the helix-coil conversion of tRNA^Phe may be approximately described by the model of an all-or-none process.     

Interaction of manganese with fragments, complementary fragment recombinations, and whole molecules of yeast phenylalanine specific transfer RNA

Binding of Mn²⁺ to the whole molecule, fragments and complementary fragment recombinations of yeast tRNA^Phe, and to synthetic polynucleotides was studied by equilibrium dialysis. The comparison of the binding patterns of the fragments, fragment recombinations and synthetic polynucleotides with that of intact tRNA^Phe permits reasonable conclusions concerning the nature and location of the various classes of sites on tRNA^Phe. Binding of Mn²⁺ to intact tRNA^Phe consists of a co-operative and a non-co-operative phase. There are about 17 “strong” sites and several “weak” ones. Five of the 17 strong sites are associated with the co-operative phase. This phase is completely lacking in the binding of Mn²⁺ to tRNA^Phe fragments (5′-$\text{1}\text{2}$, 3′-$\text{1}\text{2}$, 5′-$\text{3}\text{5}$, 3′-$\text{2}\text{5}$), poly-(A):poly(U) and poly(I):poly(C) helices, and single stranded poly(A) and poly(U). This argues that the co-operative sites arise from the tRNA tertiary structure. This conclusion is further strengthened by the observation that cooperativity is present in a tRNA^Phe molecule which has been split in the anticodon loop, but it is absent in one which has been split in the extra loop. It is in the vicinity of the latter loop, but not the former, that tertiary interactions are seen in the crystal structure. The remaining 12 strong sites are “independent” and appear to be associated with cloverleaf helical sections.     

Half molecules of serine-specific transfer ribonucleic acids from yeast

The preparation and analysis of half molecules from tRNA^Ser are described. Two pG-halves were isolated which differed only in the presence or absence of an acetyl group on the cytidylic acid residue at position 12. The CCA-half derived from tRNA₁^Ser was isolated pure, while the CCA-half derived from tRNA₂^Ser was isolated as a mixture with the CCA-half from tRNA₁^Ser from which the terminal CpCpA had been cleaved off.The acceptor activity of the combined complementary half molecules was 90% of the one of intact tRNA^Ser. The Michaelis constant and maximal velocity of amino-acylation were found to be identical for tRNA^Ser and the combined fragments.When half molecules were present at different ratios in aminoacylation studies it was found that one pG-half molecule can mediate the charging of several CCA-half molecules. There are indications that the CCA-half molecule alone can accept some serine. The CCA-half molecule alone can be aminoacylated to a rather high degree in the presence of an excess of tRNA_ox^Ser or tRNA^Ser-a and to a small degree in the presence of tRNA_ox^Ala (yeast) but not at all in the presence of tRNA_ox^Phe or tRNA_ox^Val (E. coli).Combinations of half molecules from tRNA^Ser with the opposite half molecules from tRNA^Phe could not be aminoacylated with Ser or Phe or 15 other amino acids although one of the combinations was well associated according to gel electrophoresis and differential melting curves.     

In vitro incorporation of 2′-deoxyadenosine and 3′-deoxyadenosine into yeast tRNAPhe using tRNA nucleotidyl transferase,and properties of tRNAPhe-C-C-2′dA and tRNAPhe-C-C-3′dA

2′-Deoxyadenosine and 3′-deoxyadenosine (cordycepin) can be incorporated into the 3′-terminal position of tRNA^Phe by tRNA nucleotidyl transferase. tRNA^Phe-C-C-2′dA and tRNA^Phe-C-C-3′dA, missing the cis-diol group at the 3′-terminal end are resistant to periodate oxidation and are not able to form borate complexes. In aminoacylation experiments only the tRNA^Phe-C-C-3′dA proved to be chargeable.     

Age-dependent changes in the specificity of tRNA methyltransferases in the cerebellum of the icteric and nonicteric Gunn rat

The activity of tRNA methyltransferases present in the cerebellum of 6- and 21-day-old nonicteric and icteric Gunn rats was compared using purifiedE. coli tRNAs as substrates. At 6 days the tRNA methyltransferases of the icteric animals were significantly more effective in methylating tRNA^Glu ₂ and tRNA^Phe than were those of their nonicteric counterparts. This relationship reversed itself at 21 days. The action of the tRNA methyltransferases from the 6-day-old icteric animals led to higher proportions of 1-methyladenine in tRNA^Glu ₂ and tRNA^Phe than were obtained using the corresponding enzymes of the nonicteric animals. The proportion ofN ²-methylguanine was also higher, yet only in tRNA^fMet and not in tRNA^Phe. The study reveals much more extensive fluctuations in the activity and in the substrate recognition specificity among the cerebellar tRNA methyltransferases of the icteric than among those of the nonicteric controls during the crucial 6–21 day period of cerebellar development.