Modeling of the mechanism of interactions of oligonucleotides with 3'-end of the yeast tRNA(Phe)

Three dimensional atomic models of complexes between 10-, 15-mer long oligonucleotides and east tRNAPhe have been calculated. It has been found that the fast-forming primary complexes are the major groove complexes with the coaxial acceptor- and T-steams of the tRNA(Phe). Oligonucleotide forms a triplex of the recombinant R-triplex type. The long steams allow to make a "strong complexes" whith oligonucleotide, which delivers its 3'-end nucleotides to the vicinity of the T-loop, adjacent to the steam. These nucleotides destabilize the loop structure and initiate conformational rearrangement with a local destruction of the tRNA(Phe) and formation of the final tRNA(Phe)-oligonucleotide complementary complex. The primary complex formation and following destruction of the tRNA(Phe) constitutes the mechanism of the 'molecular wedge'. The effective anticense oligonucleotide should consist of the three segments: 1--complex initiator, 2--complex formative, 3--loop destructor an have to be complementary to the tRNA structure element of [(free end)/loop-steam-loop].     

Hybridization of antisense oligonucleotides with the 3'part of tRNA(Phe)

The interaction of antisense oligodeoxyribonucleotides with yeast tRNA(Phe) was investigated. 14-15-mers complementary to the 3'-terminal sequence including the ACCA end bind to the tRNA under physiological conditions. At low oligonucleotide concentrations the binding occurs at the unique complementary site. At higher oligonucleotide concentrations, the second oligonucleotide molecule binds to the complex due to non-perfect duplex formation in the T-loop stabilized by stacking between the two bound oligonucleotides. In these complexes the acceptor stem is open and the 5'-terminal sequence of the tRNA is accessible for binding of a complementary oligonucleotide. The results prove that the efficient binding of oligonucleotides to the 3'-terminal sequence of the tRNA occurs through initial binding to the single-stranded sequence ACCA followed by invasion in the acceptor stem and strand displacement.     

Invasion of strongly binding oligonucleotides into tRNA structure

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).     

Interaction of ribo- and deoxyriboanalogs of yeast tRNA(Phe) anticodon arm with programmed small ribosomal subunits of Escherichia coli and rabbit liver.

A synthetic ribooligonucleotide, r(CCAGACUGm-AAGAUCUGG), corresponding to the unmodified yeast tRNA(Phe) anticodon arm is shown to bind to poly(U) programmed small ribosomal subunits of both E. coli and rabbit liver with affinity two order less than that of a natural anticodon arm. Its deoxyriboanalogs d(CCAGACTGAAGATCTGG) and d(CCAGA)r(CUGm-AAGA)d(TCTGG), are used to study the influence of sugar-phosphate modification on the interaction of tRNA with programmed small ribosomal subunits. The deoxyribooligonucleotide is shown to adopt a hairpin structure. Nevertheless, as well as oligonucleotide with deoxyriboses in stem region, it is not able to bind to 30S or 40S ribosomal subunits in the presence of ribo-(poly(U] or deoxyribo-(poly (dT) template. The deoxyribooligonucleotide also has no inhibitory effect on tRNA(Phe) binding to 30S ribosomes at 10-fold excess over tRNA. Neomycin does not influence binding of tRNA anticodon arm analogs used. Complete tRNA molecule and natural modifications of anticodon arm are considered to stabilize the arm structure needed for its interaction with a programmed ribosome.     

Sequence studies of nonradioactive Mycoplasma tRNAPhe with the aid of polynucleotide phosphorylase and polynucleotide kinase

The known methods of enzymatic phosphorylation with [(32)P]phosphate of the 3'- or 5'-hydroxyl group of an oligonucleotide have been applied to oligonucleotides derived from Mycoplasma tRNA(Phe). The fingerprints obtained by both methods are very similar to each other and to that of uniformly labelled tRNA. The sequence of some oligonucleotides was determined by partial digestion of the 3'-phosphorylated fragment with spleen phosphodiesterase and of the corresponding 5'-phosphorylated fragment with venom phosphodiesterase.     

Highly efficient site-directed RNA cleavage by imidazole-containing conjugates of antisense oligonucleotides

A method has been suggested for the synthesis of conjugates of oligodeoxyribonucleotides with chemical constructs mimicking ribonuclease A active center for directed fragmentation of RNA. The method is based on the sequential addition of linker group, 9-(methylamino)anthracene, to 5' or 3' terminal phosphate of oligonucleotide and then imidazole-containing construct by cycloaddition reaction. The conjugates of oligonucleotides complementary to regions 44-61 (2B-R) and 60-76 (1C-R) of yeast phenylalanine tRNA demonstrated ability to cleave tRNA(Phe) under physiological conditions preferably at the sole phosphodiester bond (C63-A64 for 2B-R and C56-G57 for 1C-R, respectively). The half-time of tRNA(Phe) hydrolysis in the presence of 2B-R conjugate was 30 min at 2B-R concentration of 10 microM and several minutes at conjugate concentration of 50 microM.     

Conformational change of the L-shaped tRNA(Phe) molecule

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(2Glu), 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(2Glu) is significantly smaller. Further, using a fluorescent probe of 4-bromomethyl-7-methoxycoumarin introduced onto pseudouridine residue psi 55 in the T psi C loop of tRNA(Phe), Stern-Volmer quenching experiments for the probe with or without added tRNA(2Glu) were carried out. The results showed greater access of the probe to the quencher with added tRNA(2Glu). These results suggest that both arms of the L-shaped tRNA structure tend to bend inside with binding of tRNA(2Glu) and some structural collapse occurs at the corner of the L-shaped structure.     

Conformational change in the 16S rRNA in the Escherichia coli 70S ribosome induced by P/P- and P/E-site tRNAPhe binding

The effects of P/P- and P/E-site tRNA(Phe) binding on the 16S rRNA structure in the Escherichia coli 70S ribosome were investigated using UV cross-linking. The identity and frequency of 16S rRNA intramolecular cross-links were determined in the presence of deacyl-tRNA(Phe) or N-acetyl-Phe-tRNA(Phe) using poly(U) or an mRNA analogue containing a single Phe codon. For N-acetyl-Phe-tRNA(Phe) with either poly(U) or the mRNA analogue, the frequency of an intramolecular cross-link C967 x C1400 in the 16S rRNA was decreased in proportion to the binding stoichiometry of the tRNA. A proportional effect was true also for deacyl-tRNA(Phe) with poly(U), but the decrease in the C967 x C1400 frequency was less than the tRNA binding stoichiometry with the mRNA analogue. The inhibition of the C967 x C1400 cross-link was similar in buffers with, or without, polyamines. The exclusive participation of C967 with C1400 in the cross-link was confirmed by RNA sequencing. One intermolecular cross-link, 16S rRNA (C1400) to tRNA(Phe)(U33), was made with either poly(U) or the mRNA analogue. These results indicate a limited structural change in the small subunit around C967 and C1400 during tRNA P-site binding sensitive to the type of mRNA that is used. The absence of the C967 x C1400 cross-link in 70S ribosome complexes with tRNA is consistent with the 30S and 70S crystal structures, which contain tRNA or tRNA analogues; the occurrence of the cross-link indicates an alternative arrangement in this region in empty ribosomes.     

Interaction of human and Escherichia coli tRNA(Phe) with human 80S ribosomes in the presence of oligo- and polyuridylate templates.

Human placenta and Escherichia coli Phe-tRNA(Phe) and N-AcPhe-tRNA(Phe) binding to human placenta 80S ribosomes was studied at 13 mM Mg2+ and 20 degrees C in the presence of poly(U), (pU)6 or without a template. Binding properties of both tRNA species were studied. Poly(U)-programmed 80S ribosomes were able to bind charged tRNA at A and P sites simultaneously under saturating conditions resulting in effective dipeptide formation in the case of Phe-tRNA(Phe). Affinities of both forms of tRNA(Phe) to the P site were similar (about 1 x 10(7) M-1) and exceeded those to the A site. Affinity of the deacylated tRNA(Phe) to the P site was much higher (association constant > 10(10) M-1). Binding at the E site (introduced into the 80S ribosome by its 60S subunit) was specific for deacylated tRNA(Phe). The association constant of this tRNA to the E site when A and P sites were preoccupied with N-AcPhe-tRNA(Phe) was estimated as (1.7 +/- 0.1) x 10(6) M-1. In the presence of (pU)6, charged tRNA(Phe) bound loosely at the A and P sites, and the transpeptidation level exceeded the binding level due to the exchange with free tRNA from solution. Affinities of aminoacyl-tRNA to the A and P sites in the presence of (pU)6 seem to be the same and much lower than those in the case of poly(U). Without a messenger, binding of the charged tRNA(Phe) to 80S ribosomes was undetectable, although an effective transpeptidation was observed suggesting a very labile binding of the tRNA simultaneously at the A and P sites.     

Effect of hairpin DNA on poly(phenylalanine) synthesis.

In a poly(U) dependent poly(Phe) synthesis, an unaminoacylated tRNA(phe) binds to a ribosome and inhibits poly(Phe) synthesis in excess tRNA(Phe). A small DNA fragment having a hairpin structure was added to this system and was found to prevent an unaminoacylated tRNA(phe) from binding to a ribosome and enhance the efficiency of poly(Phe) synthesis in the presence of excess tRNA(phe).     

Phenylalanyl-tRNA synthetase of Escherichia coli K 10. Multiple enzyme-aminoacyl-tRNA complexes as a consequence of substrate specificity.

The interaction between Phe-tRNA(Phe) or other acyl-tRNA derivatives thereof and phenylalanyl-tRNA synthetase of Escherichia coli K 10 has been investigated by nonequilibrium dialysis, by fluorescence titration in the presence of 2-p-toluidinylnaphthalene-6-sulfonate, by the kinetics of the aminoacylation of tRNA(Phe), and by the kinetics of the catalytic hydrolysis of Phe-tRNA(Phe). Phe-tRNA(Phe), or derivatives thereof, forms two types of complexes with the synthetase. One type involves the attachment of the phenylalanyl moiety to the phenylalanine-specific site of the enzyme, and the other type, to the tRNA(Phe)-specific binding site. They resemble alternative modes of a destabilized enzyme-product complex and are predicted on the basis of thermodynamic considerations. The two modes of binding of acyl-tRNA compete with each other. The attachment of Phe-tRNA(Phe) to the phenylalanine-specific site dominates. At equilibrium, this complex is present at a fourfold higher concentration than the other type of complex. The HNO2 deaminated Phe-tRNA(Phe) binds exclusively to the site specific for L-phenylalanine. On the contrary, Ile-tRNA(Phe) adds at 94.1% to the tRNA(Phe)-specific site. The association of Phe-tRNA(Phe) with this site leads to enzymatic hydrolysis into L-phenylalanine and tRNA(Phe). The complex involving the phenylalanine-specific site is hydrolytically unproductive. L-Phenylalanine acts as an activator of the hydrolysis by occupying the amino acid specific site and by shifting the equilibrium between the complexes toward the binding ot Phe-tRNA(Phe) at the tRNA(Phe)-specific site. The association of Phe-tRNA(Phe) at the phenylalanine-specific site does not interfere sterically with the binding of free tRNA(Phe). The sequential addition of free and aminoacylated tRNA(Phe) exhibits negative cooperativity. Such a mechanism could help to expel the product from the enzyme.     

Characterization of the lead(II)-induced cleavages in tRNAs in solution and effect of the Y-base removal in yeast tRNAPhe

The specificity of lead(II)-induced hydrolysis of yeast tRNA(Phe) was studied as a function of concentration of Pb2+ ions. The major cut was localized in the D-loop and minor cleavages were detected in the anticodon and T-loops at high metal ion concentration. The effects of pH, temperature, and urea were also analyzed, revealing a basically unchanged specificity of hydrolysis. In the isolated 5'-half-molecule of yeast tRNAPhe not cut was found in the D-loop, indicating its stringent dependence on T-D-loop interaction. Comparison of hydrolysis patterns and efficiencies observed in yeast tRNA(Phe) with those found in other tRNAs suggests that the presence of a U59-C60 sequence in the T-loop is responsible for the highly efficient and specific hydrolysis in the spatially close region of the D-loop. The efficiencies of D-loop cleavage in intact yeast tRNA(Phe) and in tRNA(Phe) deprived of the Y base next to the anticodon were also compared at various Pb2+ ion concentrations. Kinetics of the D-loop hydrolysis analyzed at 0, 25, and 37 degrees C showed a 6 times higher susceptibility of tRNA(Phe) minus Y base (tRNA(Phe)-Y) to lead(II)-induced hydrolysis than in tRNA(Phe). The observed effect is discussed in terms of a long-distance conformational transition in the region of the interacting D- and T-loops triggered by the Y-base excision.