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PubMed1981

tRNA synthesis: identification of in vivo precursor tRNAs from parental and mutant yeast strains.

In vivo yeast precursor tRNAs have been identified using a modification of the Northern-hybridization procedure. Two species of pre-tRNA Tyr, 1 species of pre-tRNA Ser2 and 2 species of pre-tRNA Serminor have been found in all yeast strains examined, including parental strains and strains harboring mutations affecting tRNA function. One of the tRNA Tyr strains harboring and one of the pre-tRNA SerUCG are the same size as the unspliced pre-tRNAs which accumulate in the yeast mutant rna1. The in vivo tRNA Tyr precursors detected in these studies also appear similar with the RNA species identified when cloned yeast tRNA Tyr is transcribed and processed by Xenopus oocytes and/or Xenopus extracts. We have also studied the precursor and mature tRNA Tyr species from 22 mutants which contain mutations in the SUP4 tyrosine-inserting suppressor locus. The RNA from 2 mutants mapping at the G52 position showing an aberrantly migrating "mature" tRNA Tyr. Although several of those cloned mutant genes showed transcript products of altered size in in vitro transcription studies (1), we did not detect such altered transcripts in vivo.

Animals↗
PubMed1986

Structural homology between elongation factors EF-Tu from Bacillus stearothermophilus and Escherichia coli in the binding site for aminoacyl-tRNA.

Elongation factor EF-Tu (Mr approximately equal to 50 000) and elongation factor EF-G (Mr approximately equal to 78 000) were isolated from Bacillus stearothermophilus in a homogeneous form. The ability of EF-Tu to participate in protein synthesis is rapidly inactivated by N-tosyl-L-phenyl-alanylchloromethane (Tos-PheCH2Cl). EF-Tu X GTP is more susceptible to the inhibition by Tos-PheCH2Cl than is EF-Tu X GDP. Tos-PheCH2Cl forms a covalent equimolar complex with the factor by reacting with a cysteine residue in its molecule. The labelling of EF-Tu by the reagent irreversibly destroys its ability to bind aminoacyl-tRNA, which in turn protects the protein from this inactivation. This indicates that the modification of EF-Tu by Tos-PheCH2Cl occurs at the aminoacyl-tRNA binding site of the protein. To identify and characterize the site of aminoacyl-tRNA binding in EF-Tu, the factor was labelled with [14C]Tos-PheCH2Cl, digested with trypsin, the resulting peptides were separated by high-performance liquid chromatography and the sequence of the radioactive peptide was determined. The peptide has identical structure with an Escherichia coli EF-Tu tryptic peptide comprising the residues 75-89 and the Tos-PheCH2Cl-reactive cysteine at position 81 [Jonák, J., Petersen, T. E., Clark, B. F. C. and Rychlík, I. (1982) FEBS Lett. 150, 485-488]. Experiments on photo-oxidation of EF-Tu by visible light in the presence of rose bengal dye showed that there are apparently two histidine residues in elongation factor Tu from B. stearothermophilus which are essential for the interaction with aminoacyl-tRNA. This is clearly reminiscent of a similar situation in E. coli EF-Tu [Jonák, J., Petersen, T. E., Meloun, B. and Rychlík, I. (1984) Eur. J. Biochem. 144, 295-303]. Our results provide further evidence for the conserved nature of the site of aminoacyl-tRNA binding in elongation factor EF-Tu and show that Tos-PheCH2Cl reagent might be a favourable tool for the identification of the site in the structure of prokaryotic EF-Tus.

Amino Acid Sequence↗
PubMed1975

Nucleotide sequence studies of normal and genetically altered glycine transfer ribonucleic acids from Escherichia coli.

The total nucleotide sequence of tRNAGGA/G -Gly2 from Escherichia coli is pG-C-G-G-G-C-A-U-C-G-U-A-U-A-A-U-G-G-C-U-A-U-U-A-C-C-U-C-A-G-C-C-U-N-C-C-A-A-G-C-U-G-A-U-G-A-U-G-C-G-G-G-T-psi-C-G-A-U-U-C-C-C-G-C-U-G-C-C-C-G-C-U-C-C-AOH, where T- at position 53 is ribothymidylic acid, and psi- at position 54 is pseudouridylic acid; N- at position 36 is an unidentified derivative of uridylic acid, and is present in modified form in a portion of tRNAGGA/G -Gly 2 molecules isolated from E. coli cells. The missense suppressor mutation, glyTsuA36(HA), results in a C yields U base substitution at the 3' end of the anticodon of tRNAGGA/G -Gly 2 (nucleotide position 38). A secondary effect of this base substitution is the modification of the A residue directly adjacent to the 3' end of the anticodon of tRNAsuA36(HA), -Gly 2 suggesting that the enzymes responsible for this modification recognize the anticodon sequences of prospective tRNA substrates. The creation of a missense-suppressing tRNA, tRNAsuA36(HA), -Gly 2 by an alteration of the anticodon sequence of tRNAGGA/G -Gly 2 is analogous to mechanisms whereby other suppressor tRNAs have arisen. The high degree of nucleotide sequence homology between the amino acid acceptor stems and anticodon regions of four glycine isoaccepting tRNAs specified by E. coli and bacteriophage T4 suggests that these regions may be recognized by the glycyl-tRNA synthetase; the involvement of the anticodon region in the synthetase recognition process is supported by the greatly decreased rate of aminoacylation of tRNAsuA36(HA) -Gly 2.

Anticodon↗
PubMed1983

Chemical modification in situ of Escherichia coli 30 S ribosomal proteins by the site-specific reagent pyridoxal phosphate. Inactivation of the aminoacyl-tRNA and mRNA binding sites.

epsilon-Amino groups of lysines of 30 S ribosomal subunits with affinity for phosphate groups were selectively modified in situ by reaction with pyridoxal phosphate and reduction of the Schiff base with nonradioactive or radioactive sodium borohydride. This reaction modified only a limited number of ribosomal proteins and resulted in the loss of only some 30 S activities. The modified proteins were identified and the extent of their modification determined. The main targets of the reaction were S3 greater than S1 greater than S6. The activity most severely affected by the pyridoxal phosphate reaction was mRNA-dependent aminoacyl-tRNA binding. Some inhibition of poly(U) binding was also observed, while neither binding of initiation factors nor association with 50 S subunits was inhibited. The inhibition of aminoacyl-tRNA binding showed distinct selectivity: the inhibition was far greater with NAcPhe-tRNA than with fMet-tRNA and with "A" site than with "P" site binding. In addition, initiation complex formation with some mRNAs (e.g. MS2 RNA) was affected more than with others (e.g. T7 early mRNA). Ribosome reconstitution experiments showed that the modification of protein S3 was the primary cause of the inhibition; a role was also played by ribosomal proteins S1, S2, and S21. Substrate protection experiments showed that the 30 S activity can be protected from pyridoxal phosphate inactivation upon formation of a ternary complex with poly(U) and tRNAPhe or NAcPhe-tRNAPhe. Accordingly, the extent of modification of ribosomal protein S3 was reduced in the ternary complex while modification of S1 was reduced in the presence of poly(U) alone.

Escherichia coli↗
PubMed1976

Further evidence for the participation of proteins S 3, S 14 and S 19 in tRNA binding to E. coli 30 S subunits.

Previous studies have shown that iodination of 30 S subunits causes inactivation for both enzymatic fMet-tRNA and non-enzymatic phe-tRNA binding activities. This inactivation was shown to be due to the modification of three to five ribosomal proteins [1]. In this report the role of these proteins in tRNA binding activity has been further studied. Purified ribosomal proteins, isolated from modified subunits, are re-assembled into otherwise unmodified 30 S ribosomes and assayed for tRNA binding capacity. The presence of modified S 3, S 14 and S 19 (S 15) in the reconstituted particle results in substantial reduction of both fMet-tRNA and phe-tRNA binding activities. This reduction in tRNA binding activity does not appear to be due to an assembly defect.

Binding Sites↗
PubMed2000

Hypermodified nucleosides in the anticodon of tRNALys stabilize a canonical U-turn structure.

Modified nucleosides in the anticodon domain of Escherichia coli tRNA(Lys) are necessary for high-affinity codon recognition and reading frame maintenance. Human tRNA(Lys,3) is the specific primer for HIV-1 reverse transcriptase and also requires nucleoside modification for proper function. We now present NMR solution structures for the fully modified 17-nucleotide E. coli tRNA(Lys) anticodon stem-loop domain (ASL). NMR data were also collected for several partially modified ASLs, revealing the contributions each modified nucleoside (mnm(5)s(2)U34, t(6)A37, and psi39) makes in transforming the disordered, unmodified tRNA ASL into the highly ordered native structure. The solution structure of the native ASL domain provides insight into longstanding questions regarding both wobble position modification and the nearly ubiquitous t(6)A37 found in tRNAs with an adjacent U at position 36. Native tRNA(Lys) has a U-turn structure similar to the yeast tRNA(Phe) crystal structure, unlike previously proposed "unconventional" anticodon structures characterized by stable interactions between mnm(5)s(2)U-34 and t(6)A-37.

Anticodon↗
PubMed1997

Interaction of N-tosyl-L-phenylalanylchloromethane with Thermus thermophilus elongation factor Tu.

The interaction of N-tosyl-L-phenylalanylchloromethane (TosPheCH2Cl) with Thermus thermophilus elongation factor Tu (EF-Tu) was studied by affinity labelling and NMR spectroscopy. TosPheCH2Cl binds to GDP and GTP conformers of EF-Tu. The interaction of TosPheCH2Cl with EF-Tu x GDP leads to alkylation of Cys82, while interaction of TosPheCH2Cl with EF-Tu x GTP does not lead to covalent labelling. [A82]EF-Tu, in which the Cys82 is replaced by Ala, has similar properties to wild-type EF-Tu with respect to GTPase activity, binding of guanine nucleotides, interaction with elongation factor Ts (EF-Ts) and interaction with ribosomes. This structural change did not lead to changes, compared with wild-type EF-Tu in the functionality of [A82]EF-Tu, either in the GTP or in the GDP conformation. TosPheCH2Cl binds to EF-Tu x GTP with a dissociation constant of 10 microM. The interaction of TosPheCH2Cl with EF-Tu promotes the hydration of the carbonyl group of TosPheCH2Cl. TosPheCH2Cl competes with aminoacyl-tRNA for its binding site on EF-Tu x GTP. Covalent modification of Cys82 by TosPheCH2Cl does not prevent nucleotide binding and GTPase activity, but interferes with the interaction with aminoacyl-tRNA. TosPheCH2Cl probably mimics the aminoacyl residue of the aminoacyl-tRNA and binds to its binding site on EF-Tu x GTP. This rather specific interaction with EF-Tu x GTP does not allow the modification of Cys82, whereas the loose interaction of TosPheCH2Cl with EF-Tu x GDP leads to alkylation of this residue.

Affinity Labels↗
PubMed1990

Ternary complexes of Escherichia coli aminoacyl-tRNAs with the elongation factor Tu and GTP: thermodynamic and structural studies.

The interaction of 18 different Escherichia coli aminoacyl-tRNA species with elongation factor Tu and GTP has been measured by a fluorescence titration assay under equilibrium conditions. The dissociation constants range from 1.9 +/- 0.2.10(-10) M up to 1020 +/- 250.10(-10) M depending on the nucleotide sequence, secondary structure and the chemical composition of the aminoacyl residue of the particular aminoacyl-tRNA. The 'aminoacyl domain' of tRNA consisting of the single stranded, four-nucleotide-long 3'-terminus, aminoacyl stem of seven base-pairs, T-stem and T-loop contains all elements necessary for binding EF-Tu.GTP. The efficiency of aminoacyl-tRNA interaction with EF-Tu.GTP is modulated by the sequence of this 'aminoacyl domain' and by natural modification of its nucleotide residues. An oligoribonucleotide resembling the aminoacyl stem of E.coli tRNA(Ala) and consisting of a four-membered 3'-end, a stem of seven base-pairs and a loop of six nucleotides was prepared by total chemical synthesis on a polymer support. It can be enzymatically aminoacylated by alanine but does not bind in its aminoacylated form to EF-Tu.GTP.

Anticodon↗
PubMed2004

A novel method for sequence placement of modified nucleotides in mixtures of transfer RNA.

Sequence placement of post-transcriptionally modified nucleosides in tRNA can be experimentally difficult, particularly in cases involving new or unexpected modifications or sequence sites. We describe a mass spectrometry-based approach to this problem, involving the following steps: crude isolations of one or several tRNAs by HPLC from an unfractionated tRNA mixture; digestion to oligonucleotide mixtures by RNase T1; analysis by combined HPLC/electrospray ionization-MS for recognition of modifications; and direct gas-phase sequencing of selected targets in the mixture by LC/MS/MS. Isoacceptor identity can be established in favorable cases when tRNA gene sequences are available.

Chromatography, High Pressure Liquid↗
PubMed1990

A potato mitochondrial isoleucine tRNA is coded for by a mitochondrial gene possessing a methionine anticodon.

The nucleotide sequence of a potato mitochondrial tRNA(Ile) has been determined. This tRNA has a lysidine-like minor nucleotide in the first position of the anticodon (position 34). The corresponding mitochondrial gene has been cloned and sequenced. This gene has a CAT anticodon which corresponds to methionine. The C residue in the anticodon must therefore be post-transcriptionally modified. The mature tRNA has isoleucine-accepting activity but no methionine-accepting activity. This is the first report showing that in plant mitochondria a post-transcriptional modification could change the amino-acid specificity of a tRNA, as compared to that of the corresponding gene.

Anticodon↗
PubMed1999

Translational regulation by modifications of the elongation factor Tu.

EF-Tu from E. coli, one of the superfamily of GTPase switch proteins, plays a central role in the fast and accurate delivery of aminoacyl-tRNAs to the translating ribosome. An overview is given about the regulatory effects of methylation, phosphorylation and phage-induced cleavage of EF-Tu on its function. During exponential growth, EF-Tu becomes monomethylated at Lys56 which is converted to Me2Lys upon entering the stationary phase. Lys56 is in the GTPase switch-1 region (residues 49-62), a strongly conserved site involved in interactions with the nucleotide and the 5' end of tRNA. Methylation was found to attenuate GTP hydrolysis and may thus enhance translational accuracy. In vivo 5-10% of EF-Tu is phosphorylated at Thr382 by a ribosome-associated kinase. In EF-Tu-GTP, Thr382 in domain 3 has a strategic position in the interface with domain 1; it is hydrogen-bonded to Glu117 that takes part in the switch-2 mechanism, and is close to the T-stem binding site of the tRNA, in a region known for many kirromycin-resistance mutations. Phosphorylation is enhanced by EF-Ts, but inhibited by kirromycin. In reverse, phosphorylated EF-Tu has an increased affinity for EF-Ts, does not bind kirromycin and can no longer bind aminoacyi tRNA. The in vivo role of this reversible modification is still a matter of speculation. T4 infection of E. coli may trigger a phase-exclusion mechanism by activation of Lit, a host-encoded proteinase. As a result, EF-Tu is cleaved site-specifically between Gly59-Ile60 in the switch-1 region. Translation was found to drop beyond a minimum level. Interestingly, the identical sequence in the related EF-G appeared to remain fully intact. Although the Lit cleavage-mechanism may eventually lead to programmed cell death, the very efficient prevention of phage multiplication may be caused by a novel mechanism of in cis inhibition of late T4 mRNA translation.

Escherichia coli↗
PubMed1990

The three cysteine residues of cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae are not essential for its activity.

Cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae is a dimer made up of identical subunits (Mr 63,000) each of these containing three cysteines (residues 255, 512 and 519 in the amino acid sequence). Thiol-specific probes were used to label these cysteines and study the resulting effect of the modification on the kinetic parameters of both the ATP/PPi exchange and tRNA aminoacylation reactions. Using the classical techniques of protein chemistry it was shown that none of the three cysteines was labelled with iodoacetic acid, whilst N-ethylmaleimide and 5,5'-dithiobis(2-nitrobenzoate) reacted with Cys512 and Cys255, respectively. Only the latter modification was accompanied by a decrease in the rates of both enzyme activities whilst the Km values for the various substrates remained unaffected. Site-directed mutagenesis was also used to replace each of the three cysteines by other residues, either individually or simultaneously. For these experiments the enzyme was expressed in Escherichia coli using an expression vector bearing the structural gene in which the first 13 codons were replaced by the first 14 of the CII lambda gene. The resulting substitution in the amino-terminal part of the expressed enzyme had no effect on the kinetic parameters, compared to those of the enzyme purified from S. cerevisiae. Taking into account the consequences of such substitutions, as well as those of chemical modifications on the two reactions catalysed by the enzyme. ATP/PPi exchange and tRNA aminoacylation, it could be concluded that none of these three cysteines plays any essential role in either substrate binding or catalysis.

Adenosine Triphosphate↗
PubMed1976

Changes in transfer ribonucleic acids of Bacillus subtilis during different growth phases.

The transfer ribonucleic acids (tRNAs) of B. subtilis at different growth phases are examined for changes in the composition and the methylation of minor constituents. The composition of the tRNAs indicates about equal amounts of adenosine and uridine, and of guanosine and cytidine. About 3-4 residues are present as modified bases in the average tRNA molecule. The net composition of tRNAs appears to remain unaltered during different growth phases. In vitro methylation of tRNAs indicates lack of methyl groups in both exponentially growing cells and spores. In vivo methylation studies show tRNA methylation occurs during the stationary phase in the absence of net tRNA synthesis. Thus, both in vitro and in vivo methylation indicate that the tRNAs in exponentially growing cells do not contain their full complement of modified bases. More complete modification is noted in tRNAs from stationary cells or spores. Hence, tRNA mofifications in general are preserved with fidelity even in the dormant spore but the possibility is left open that specific modifications of selected isoacceptors of tRNAs may occur.

Bacillus subtilis↗
PubMed1994

Selective perturbation of G530 of 16 S rRNA by translational miscoding agents and a streptomycin-dependence mutation in protein S12.

Previous studies have shown that a concise set of universally conserved bases in 16 S rRNA are strongly protected from attack by chemical probes when tRNA is bound specifically to the ribosomal A site. Two of these bases, A1492 and A1493, are located in the cleft of the 30 S subunit, the site of codon-anticodon interaction. A third residue, G530, is located within the highly conserved 530 stem-loop, a region that is involved in interactions with proteins S4 and S12, mutations in which perturb the translational error frequency. The 530 loop is also thought to be located at or near the site of interaction of elongation factor Tu on the 30 S subunit, a location that is distinct from the decoding site. This study monitors the response of these two A-site-related regions of 16 S rRNA to a variety of translational miscoding agents. Several of these agents, including streptomycin, neomycin and ethanol, selectively potentiate tRNA-dependent protection of residue G530 from kethoxal modification; in contrast, little change in reactivity of residues A1492 and A1493 is observed. These results are consistent with the previously demonstrated importance of G530 for A-site function and, moreover, suggest a common mechanism of action for these miscoding agents, even though they appear to have distinctly different modes of interaction with 16 S rRNA. In contrast to the miscoding agents, we find that a streptomycin-dependence (SmD) mutation in protein S12, which causes ribosomes to be hyperaccurate, antagonizes tRNA-dependent protection of G530. The possibility that 5' or 3' flanking regions of mRNA could be involved in tRNA-dependent protection of G530 was tested by using different lengths of oligo(U) to promote binding of tRNA(Phe) to the A site. The relative levels of protection of G530, A1492 and A1493 were unchanged as the size of the mRNA fragment was decreased from 16 to 6 bases in length. We conclude, therefore, that for protection of G530 to be the result of direct contact with message, it must necessarily be located directly at the decoding site; otherwise, its protection is best explained by allosteric interactions, either with mRNA, or with the codon-anticodon complex. These results are discussed in terms of a model wherein the conformation of the 530 loop is correlated with the affinity of the ribosome for elongation factor Tu.

Base Sequence↗
PubMed1999

UV-induced modifications in the peptidyl transferase loop of 23S rRNA dependent on binding of the streptogramin B antibiotic, pristinamycin IA.

The naturally occurring streptogramin B antibiotic, pristinamycin IA, which inhibits peptide elongation, can produce two modifications in 23S rRNA when bound to the Escherichia coli 70S ribosome and irradiated at 365 nm. Both drug-induced effects map to highly conserved nucleotides within the functionally important peptidyl transferase loop of 23S rRNA at positions m2A2503/psi2504 and G2061/A2062. The modification yields are influenced strongly, and differentially, by P-site-bound tRNA and strongly by some of the peptidyl transferase antibiotics tested, with chloramphenicol producing a shift in the latter modification to A2062/C2063. Pristinamycin IA can also produce a modification on binding to deproteinized, mature 23S rRNA, at position U2500/C2501. The same modification occurs on an approximately 37-nt fragment, encompassing positions approximately 2496-2532 of the peptidyl transferase loop that was excised from the mature rRNA using RNAse H. In contrast, no antibiotic-induced effects were observed on in vitro T7 transcripts of full-length 23S rRNA, domain V, or on a fragment extending from positions approximately 2496-2566, which indicates that one or more posttranscriptional modifications within the sequence Cm-C-U-C-G-m2A-psi-G2505 are important for pristinamycin IA binding and/or the antibiotic-dependent modification of 23S rRNA.

Anti-Bacterial Agents↗
PubMed1993

tRNA-guanine transglycosylase from Escherichia coli. Overexpression, purification and quaternary structure.

tRNA-guanine transglycosylase (TGT) is the enzyme responsible for the posttranscriptional modification of specific tRNAs (Asn, Asp, His and Tyr) with queuine. In E. coli this modification occurs via a two-step reaction: (1) TGT-catalyzed base exchange of guanosine-34 with preQ1 (7-aminomethyl-7-deazaguanine) and (2) addition of a cyclopentenediol moiety to the preQ1-34 tRNA. E. coli TGT is normally expressed at very low levels (approximately 1 mg from 500 g cells). The sequence of the queuine operon of E. coli has recently been reported by Reuter et al. (1991). We have cloned the tgt gene into an overexpressing vector in order to provide a more efficient preparation of TGT. A simple, four-step purification scheme yields 78 mg of homogeneous TGT per liter of cell culture (A600 = 5 to 6). Amino-terminal protein sequencing confirms the identity of the recombinant protein and indicates that the initiator methionine is retained in the mature form. Native-PAGE of TGT and SDS-PAGE of cross-linked TGT are most consistent with a hexameric quaternary structure for the enzyme. The cross-linking data also suggests that the enzyme exists as a dimer of trimers of identical 42.5 kDa subunits (total M(r) = 255 kDa. The enzyme is inactivated by cross-linking with the bisimidoester, dimethylsuberimidate. Substrate (tRNA) protects the enzyme against cross-linking and inactivation by dimethylsuberimidate and against inactivation by modification with ethylacetimidate, a monofunctional, imidoester. This indicates that the enzymic residues (presumably lysines) that are involved in cross-linking and the inactivation are in the active site of the enzyme.

Amino Acids↗
PubMed1995

Mutations of Arabidopsis thaliana pre-tRNA(Tyr) affecting pseudouridylation of U35.

The structural and sequence requirements for the biosynthesis of tRNA(Tyr) pseudouridine (psi 35) have been studied. Nucleotide substitution at the 32nd position slightly reduced modification efficiency in the case of transition (C32 to U32) while transversion (C32 to G32) had no effect on the modification process in wheat germ extract. Insertion of one nucleotide into the anticodon stem caused a 2-fold reduction of modification efficiency. Mutants with a partially deleted 12 nt long intron of pre-tRNA(Tyr) exhibited different effects: deletion of 5 nt (7 nt long intron) gave only a reduction in pseudouridylation while deletion of 7 nt (5 nt long intron) almost completely abolished the reaction. The generated mini-substrate consisting of pre-tRNA(Tyr) anticodon stem and intron sequence was partially modified which proved that the crucial elements for recognition of psi 35 introduction had to present in this construct.

Arabidopsis↗
PubMed1989

Modified tRNAs for probing tRNA binding sites on the ribosome.

Unusual chemical properties of hypermodified nucleosides N6-(threoninocarbonyl)adenosine (t6 A) located at position 37 and 3-(3-amino-3-carboxypropyl)uridine (acp3U) located at position 20:1 have been utilized for the introduction of photoreactive azidonitrophenyl probes to the anticodon loop and to the dihydrouridine loop of yeast tRNA(mMet) and lupin tRNA(mMet), respectively. The very efficient and selective modification procedures involve condensat on of the carboxyl group of t6A with ethylenediamine in the presence of a water soluble carbodiimide followed by acylation of the newly introduced amino group with the respective N-hydroxysuccinimide ester, and acylation of the primary amino or up of acp3U with the respective N-hydroxysuccinimide ester. Binding and crosslinking of the modified, uncharged tRNAs to E coli ribosome have been studied in the presence and absence of poly(AUG) as a message. Both tRNAs carrying about 20 A long photoreactive probes retain their binding activity and upon irradiation with visible light crosslink to the ribosome with high yields showing their usefulness for structural studies on the tRNA-ribosome complex.

Adenosine↗