Anticodon domain methylated nucleosides of yeast tRNA(Phe) are significant recognition determinants in the binding of a phage display selected peptide.

The contributions of the natural modified nucleosides to RNA identity in protein/RNA interactions are not understood. We had demonstrated that 15 amino acid long peptides could be selected from a random phage display library using the criterion of binding to a modified, rather than unmodified, anticodon domain of yeast tRNA(Phe) (ASL(Phe)). Affinity and specificity of the selected peptides for the modified ASL(Phe) have been characterized by fluorescence spectroscopy of the peptides' tryptophans. One of the peptides selected, peptide t(F)2, exhibited the highest specificity and most significant affinity for ASL(Phe) modified with 2'-O-methylated cytidine-32 and guanosine-34 (Cm(32) and Gm(34)) and 5-methylated cytidine-40 (m(5)C(40)) (K(d) = 1.3 +/- 0.4 microM) and a doubly modified ASL(Phe)-Gm(34),m(5)C(40) and native yeast tRNA(Phe) (K(d) congruent with 2.3 and 3.8 microM, respectively) in comparison to that for the unmodified ASL(Phe) (K(d) = 70.1 +/- 12.3 microM). Affinity was reduced when a modification altered the ASL loop structure, and binding was negated by modifications that disfavored hairpin formation. Peptide t(F)2's higher affinity for the ASL(Phe)-Cm(32),Gm(34),m(5)C(40) hairpin and fluorescence resonance energy transfer from its tryptophan to the hypermodified wybutosine-37 in the native tRNA(Phe) placed the peptide across the anticodon loop and onto the 3'-side of the stem. Inhibition of purified yeast phenylalanyl-tRNA synthetase (FRS) catalyzed aminoacylation of cognate yeast tRNA(Phe) corroborated the peptide's binding to the anticodon domain. The phage-selected peptide t(F)2 has three of the four amino acids crucial to G(34) recognition by the beta-structure of the anticodon-binding domain of Thermus thermophilus FRS and exhibited circular dichroism spectral properties characteristic of beta-structure. Thus, modifications as simple as methylations contribute identity elements that a selected peptide specifically recognizes in binding synthetic and native tRNA and in inhibiting tRNA aminoacylation.     

The inhibitory effect of oligodeoxynucleotides complementary to different fragments of tRNA structure on the enzymatic binding of Phe-tRNA to poly-U-programmed eukaryotic ribosomes

The effect of several oligodeoxynucleotides complementary to the fragments of yellow lupin tRNA(Phe) was tested in the aminoacylation of tRNA(Phe) and in the binding of Phe-tRNA(Phe) to poly-U-programmed eukaryotic ribosomes. Oligonucleotides tested in the aminoacylation test did not give any inhibition. Monomers and dimers did not have any significant influence on the binding assay, either. A different percentage of inhibition of the binding of Phe-tRNA to ribosomes has been observed for oligonucleotides. Heptamer complementary to the anticodon loop gave 100% inhibition of the binding reaction. However, the oligonucleotides complementary to both the anticodon loop and stem and longer than the heptamer were much less effective inhibitors. A high inhibitory effect was also observed for trimers and for the decamer complementary to the D-loop and CCA-end.     

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.     

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.     

Role of modified nucleosides of yeast tRNAPhe in ribosomal binding

Naturally occurring nucleoside modifications are an intrinsic feature of transfer RNA (tRNA), and have been implicated in the efficiency, as well as accuracy-of codon recognition. The structural and functional contributions of the modified nucleosides in the yeast tRNA(Phe) anticodon domain were examined. Modified nucleosides were site-selectively incorporated, individually and in combinations, into the heptadecamer anticodon stem and loop domain, (ASL(Phe)). The stem modification, 5-methylcytidine, improved RNA thermal stability, but had a deleterious effect on ribosomal binding. In contrast, the loop modification, 1-methylguanosine, enhanced ribosome binding, but dramatically decreased thermal stability. With multiple modifications present, the global ASL stability was mostly the result of the individual contributions to the stem plus that to the loop. The effect of modification on ribosomal binding was not predictable from thermodynamic contributions or location in the stem or loop. With 4/5 modifications in the ASL, ribosomal binding was comparable to that of the unmodified ASL. Therefore, modifications of the yeast tRNA(Phe) anticodon domain may have more to do with accuracy of codon reading than with affinity of this tRNA for the ribosomal P-site. In addition, we have used the approach of site-selective incorporation of specific nucleoside modifications to identify 2'O-methylation of guanosine at wobble position 34 (Gm34) as being responsible for the characteristically enhanced chemical reactivity of C1400 in Escherichia coli 16S rRNA upon ribosomal footprinting of yeast tRNA(Phe). Thus, effective ribosome binding of tRNA(Phe) is a combination of anticodon stem stability and the correct architecture and dynamics of the anticodon loop. Correct tRNA binding to the ribosomal P-site probably includes interaction of Gm34 with 16S rRNA C1400.     

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.     

A magnesium-induced conformational transition in the loop of a DNA analog of the yeast tRNA(Phe) anticodon is dependent on RNA-like modifications of the bases of the stem.

Two single-stranded DNA heptadecamers corresponding to the yeast tRNA(Phe) anticodon stem-loop were synthesized, and the solution structures of the oligonucleotides, d(CCAGACTGAAGATCTGG) and d(CCAGACTGAAGAU-m5C-UGG), were investigated using spectroscopic methods. The second, or modified, base sequence differs from that of DNA by RNA-like modifications at three positions; dT residues were replaced at positions 13 and 15 with dU, and the dC at position 14 with d(m5C), corresponding to positions where these nucleosides occur in tRNA(Phe). Both oligonucleotides form intramolecular structures at pH 7 in the absence of Mg2+ and undergo monophasic thermal denaturation transitions (Tm = 47 degrees C). However, in the presence of 10 mM Mg2+, the modified DNa adopted a structure that exhibited a biphasic "melting" transition (Tm values of 23 and 52 degrees C) whereas the unmodified DNA structure exhibited a monophasic denaturation (Tm = 52 degrees C). The low-temperature, Mg(2+)-dependent structural transition of the modified DNA was also detected using circular dichroism (CD) spectroscopy. No such transition was exhibited by the unmodified DNA. This transition, unique to the modified DNA, was dependent on divalent cations and occurred most efficiently with Mg2+; however, Ca2+ also stabilized the alternative conformation at low temperature. NMR studies showed that the predominant structure of the modified DNA in sodium phosphate (pH 7) buffer in the absence of Mg2+ was a hairpin containing a 7-nucleotide loop and a stem composed of 3 stable base pairs. In the Mg(2+)-stabilized conformation, the loop became a two-base turn due to the formation of two additional base pairs across the loop.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photochemical cross-linking of yeast tRNA(Phe) containing 8-azidoadenosine at positions 73 and 76 to the Escherichia coli ribosome

The 3'-terminal -A-C-C-A sequence of yeast tRNA(Phe) has been modified by replacing either adenosine-73 or adenosine-76 with the photoreactive analogue 8-azidoadenosine (8N3A). The incorporation of 8N3A into tRNA(Phe) was accomplished by ligation of 8-azidoadenosine 3',5'-bisphosphate to the 3' end of tRNA molecules which were shortened by either one or four nucleotides. Replacement of the 3'-terminal A76 with 8N3A completely blocked aminoacylation of the tRNA. In contrast, the replacement of A73 with 8N3A has virtually no effect on the aminoacylation of tRNA(Phe). Neither substitution hindered binding of the modified tRNAs to Escherichia coli ribosomes in the presence of poly(U). Photoreactive tRNA derivatives bound noncovalently to the ribosomal P site were cross-linked to the 50S subunit upon irradiation at 300 nm. Nonaminoacylated tRNA(Phe) containing 8N3A at either position 73 or position 76 cross-linked exclusively to protein L27. When N-acetylphenylalanyl-tRNA(Phe) containing 8N3A at position 73 was bound to the P site and irradiated, 23S rRNA was the main ribosomal component labeled, while smaller amounts of the tRNA were cross-linked to proteins L27 and L2. Differences in the labeling pattern of nonaminoacylated and aminoacylated tRNA(Phe) containing 8N3A in position 73 suggest that the aminoacyl moiety may play an important role in the proper positioning of the 3' end of tRNA in the ribosomal P site. More generally, the results demonstrate the utility of 8N3A-substituted tRNA probes for the specific labeling of ribosomal components at the peptidyltransferase center.     

Identification of 2'-hydroxyl groups required for interaction of a tRNA anticodon stem-loop region with the ribosome.

Synthetic RNA stem loops corresponding to positions 28-42 in the anticodon region of tRNA(Phe) bind efficiently in an mRNA-dependent manner to ribosomes, whereas those made from DNA do not. In order to identify the positions where ribose is required, the anticodon stem-loop region of tRNA(Phe) (Escherichia coli) was synthesized chemically using a mixture of 2'-hydroxyl- and 2'-deoxynucleotide phosphoramidites. Oligonucleotides whose ribose composition allowed binding were retained selectively on nitrocellulose filters via binding to 30S ribosomal subunits. The binding-competent oligonucleotides were submitted to partial alkaline hydrolysis to identify the positions that were enriched for ribose. Quantification revealed a strong preference for a 2'-hydroxyl group at position U33. This was shown directly by the 50-fold lower binding affinity of a stem loop containing a single deoxyribose at position U33. Similarly, defective binding of the corresponding U33-2'-O-methyl-substituted stem-loop RNA suggests that absence of the 2'-hydroxyl group, rather than an altered sugar pucker, is responsible. Stem-loop oligoribonucleotides from different tRNAs with U33-deoxy substitutions showed similar, although quantitatively different effects, suggesting that intramolecular rather than tRNA-ribosome interactions are affected. Because the 2'-hydroxyl group of U33 was shown to be a major determinant of the U-turn of the anticodon loop in the crystal structure of tRNA(Phe) in yeast, our finding might indicate that the U-turn conformation in the anticodon loop is required and/or maintained when the tRNA is bound to the ribosomal P site.     

Specific inactivation of Escherichia coli tRNA(Phe) by antisense DNA-treatment under Mg2+-deficient conditions

The preparation of an Escherichia coli tRNA mixture lacking several specific species may be useful for applications ranging from cell-free protein preparation to protein engineering. We have already demonstrated that tRNA(Asp) can be inactivated, or 'knocked out', with practical specificity by an antisense strategy. In the present study, we synthesized five tRNA(Phe)-targeted antisense oligonucleotides and tested if this tRNA can also be inactivated specifically. The salt conditions used previously for the tRNA(Asp) inactivation were not applicable to tRNA(Phe). Instead, Mg2+-deficient conditions were found to be useful for the inactivation of tRNAPhe by the antisense oligonucleotides. These conditions were also applicable to the inactivation of tRNA(Asp). The susceptibility to the antisense DNAs can change drastically, depending on the concentration of Mg2+.     

Enzymatic binding of aminoacyl transfer ribonucleic acid to ribosomes: the study of binding sites of 2' and 3' isomers of aminoacyl transfer ribonucleic acid.

The mechanism of enzymatic binding of AAtRNA to the acceptor site Escherichia coli ribosomes has been studied using the following aminoacyl oligonucleotides as models of the 3' terminus of AA-tRNA: C-A-Phe, C-A-(2'-Phe)H, and C-A(2'H)Phe. T-psi-C-Gp was used as a model of loop IV of tRNA. The EF-T dependent binding of Phe-tRNA to ribosomes (in the presence of either GTP or GMPPCP) and the GTPase activity associated with EF-T dependent binding of the Phe-tRNA were inhibited by C-A-Phe,C-A(2'Phe)H, and C-A(2'H)Phe. These aminoacyl oligonucleotides inhibit both the formation of ternary complex EF-Tu-GTP-AA-tRNA and the interaction of this complex with the ribosomal A site. The uncoupled EF-Tu dependent GTPase (in the absence of AA-tRNA) was also inhibited by C-A-Phe, C-A(2'Phe)H, and C-A(2'H)Phe, while nonenzymatic binding of Phe-tRNA to the ribosomal A site was inhibited by C-A-Phe and C-A(2'-Phe)H, but not by C-A(2'H)Phe. The tetranucleotide T-psi-C-Gp inhibited both enzyme binding of Phe-tRNA and EF-T dependent GTP hydrolysis. However, inhibition of the latter reaction occured at a lower concentration of T-psi-C-Gp suggesting a specific role of T-psi-C-Gp loop of AA-tRNA in the GTPase reaction. The role of the 2' and 3' isomers of AA-tRNA during enzymatic binding to ribosomes is discussed and it is suggested that 2' leads to 3' transacylation in AA-tRNA is a step which follows GTP hydrolysis but precedes peptide bond formation.     

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.     

A new type of chemically modified tRNA as a tool for the study of tRNA-aminoacyl-tRNA synthetase interaction

tRNA(Phe) in which the adenine and cytosine rings in the aminoacyl arm and in the anticodon loop were converted to alkylating derivatives by mild treatment with methyl chlorotetrolate was used to study the tRNA(Phe)-yeast phenylalanyl-tRNA(Phe) synthetase interaction. At neutral pH, modified tRNA inhibited the enzyme competitively. At pH 9 this binding is accompanied by irreversible inactivation of the enzyme due to alkylation of the alpha subunit of the synthetase. Such a derivatization of tRNA could probably be used to investigate the interaction of other tRNAs with their cognate synthetases.     

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.     

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.     

Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase: essential elements for recognition of tRNA substrates within the anticodon stem-loop

Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase (DMAPP-tRNA transferase) catalyzes the alkylation of the exocyclic amine of A37 by a dimethylallyl unit in tRNAs with an adenosine in the third anticodon position (position 36). By use of purified recombinant enzyme, steady- state kinetic studies were conducted with chemically synthesized RNA oligoribonucleotides to determine the essential elements within the tRNA anticodon stem-loop structure required for recognition by the enzyme. A 17-base oligoribonucleotide corresponding to the anticodon stem-loop of E. coli tRNA(Phe) formed a stem-loop minihelix (minihelix(Phe)) when annealed rapidly on ice, while the same molecule formed a duplex structure with a central loop when annealed slowly at higher concentrations. Both the minihelix and duplex structures gave k(cat)s similar to that for the normal substrate (full-length tRNA(Phe) unmodified at A37), although the K(m) for minihelix(Phe) was approximately 180-fold higher than full-length tRNA. The A36-A37-A38 motif, which is completely conserved in tRNAs modified by the enzyme, was found to be important for modification. Changing A36 to G in the minihelix resulted in a 260-fold reduction in k(cat) compared to minihelix(Phe) and a 13-fold increase in K(m). An A38G variant was modified with a 9-fold reduction in k(cat) and a 5-fold increase in K(m). A random coil 17-base oligoribonucleotide in which the loop sequence of E. coli tRNA(Phe) was preserved, but the 5 base pair helix stem was completely disrupted and showed no measurable activity, indicating that a helix-loop structure is essential for recognition. Finally, altering the identity of several base pairs in the helical stem did not have a major effect on catalytic efficiency, suggesting that the enzyme does not make base-specific contacts important for binding or catalysis in this region.     

Lead-catalyzed cleavage of yeast tRNAPhe mutants

Yeast tRNA(Phe) lacking modified nucleotides undergoes lead-catalyzed cleavage between nucleotides U17 and G18 at a rate very similar to that of its fully modified counterpart. The rates of cleavage for 28 tRNA(Phe) mutants were determined to define the structural requirements of this reaction. The cleavage rate was found to be very dependent on the identity and correct positioning of the two lead-coordinating pyrimidines defined by X-ray crystallography. Nucleotide changes that disrupted the tertiary interactions of tRNAPhe reduced the rate of cleavage even when they were distant from the lead binding pocket. However, nucleotide changes designed to maintain tertiary interactions showed normal rates of cleavage, thereby making the reaction of a useful probe for tRNA(Phe) structure. Certain mutants resulted in the enhancement of cleavage at a "cryptic" site at C48. The sequences of Escherichia coli tRNA(Phe) and yeast tRNA(Arg) were altered such that they acquired the ability to cleave at U17, confirming our understanding of the structural requirements for cleavage. This mutagenic analysis of the lead cleavage domain provides a useful guide for similar analysis of autocatalytic self-cleavage reactions.     

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.