An energy transfer equilibrium between two identical copies of a ribosome-bound fluorescent transfer RNA analogue: implications for the possible structure of codon-anticodon complexes.

When yeast tRNA_Pf^Phe, a derivative of tRNA^Phe in which proflavine replaces the Y base, is bound simultaneously to both the peptidyl and aminoacyl sites of a 70 S Escherichia coli ribosome, there is a rapid mutual energy transfer between the two bound tRNAs. Analysis of this energy transfer yields an upper limit for the proflavine-proflavine distance of 20 Å. It also allows an unequivocal measurement of the emission spectrum of tRNA_Pf^Phe bound at the aminoacyl site. In the presence of message this spectrum is very different from that seen in the peptidyl site, implying that in the two sites the hypermodified bases exist in significantly different environments. The rapid energy transfer leads to some loss of fluorescence anisotropy. This can be analyzed to obtain an estimate of the angle between the two proflavines: 28 ° ± 10 ° or 152 ° ± 10 °. Taken together all of these results place severe constraints on possible models of codon-anticodon complexes. The mutual energy transfer seen and analyzed on the ribosome is a convenient aspect of fluorescence spectroscopy, and it is one that should see broad application where multiple copies of a fluorescent ligand interact on a macromolecular substrate.     

The Effect of Modification of Nucleotide-37 on the Interaction of Aminoacyl-tRNA with the A Site of the 70S Ribosome

To estimate the effect of modified nucleotide 37, the interaction of two yeast aminoacyl-tRNAs (Phe-tRNA^Phe _+Y and Phe-tRNA^Phe _–Y) with the A site of complex [70S · poly(U) · deacylated tRNA^Phe in the P site] was assayed at 0–20°C. As comparisons with native Phe-tRNA^Phe _+Y showed, removal of the Y base decreased the association constant of Phe-tRNA^Phe _–Y and the complex by an order of magnitude at every temperature tested, and increased the enthalpy of their interaction by 23 kJ/mol. When the Y base was present in the anticodon loop of deacylated tRNA^Phe bound to the P site of the 70S ribosome, twice higher affinity for the A site was observed for Phe-tRNA^Phe _–Y but not for Phe-tRNA^Phe _+Y. Thus, the modified nucleotide 3" of the Phe-tRNA^Phe anticodon stabilized the codon–anticodon interaction both in the A and P sites of the 70S ribosome.     

Understanding the Sequence Specificity of tRNA Binding to Elongation Factor Tu using tRNA Mutagenesis

Measuring the binding affinities of 42 single-base-pair mutants in the acceptor and TΨC stems of Saccharomyces cerevisiae tRNA^Phe to Thermus thermophilus elongation factor Tu (EF-Tu) revealed that much of the specificity for tRNA occurs at the 49-65, 50-64, and 51-63 base pairs. Introducing the same mutations at the three positions into Escherichia coli tRNA_CAG^Leu resulted in similar changes in binding affinity. Swapping the three pairs from several E. coli tRNAs into yeast tRNA^Phe resulted in chimeras with EF-Tu binding affinities similar to those for the donor tRNA. Finally, analysis of double- and triple-base-pair mutants of tRNA^Phe showed that the thermodynamic contributions at the three sites are additive, permitting reasonably accurate prediction of the EF-Tu binding affinity for all E. coli tRNAs. Thus, it appears that the thermodynamic contributions of three base pairs in the TΨC stem primarily account for tRNA binding specificity to EF-Tu.     

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.     

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.     

Fluorescence studies of the binding of a yeast tRNAPhe derivative to Escherichia coli ribosomes.

The equilibrium binding of a highly fluorescent derivative of yeast tRNA^Phe to Escherichia coli 70 S ribosomes was studied fluorimetrically at 7 °C in 25 mm-magnesium. Under these conditions 70 S ribosomes bind two deacylated tRNAs stoichiometrically. An analysis of the binding data using a model in which occupancy of the weaker site requires prior occupancy of the stronger site leads to apparent association constants of (1.00 ± 0.05) × 10⁹m^?1 and (3.4 ± 0.2) × 10⁷m^?1. The use of an independent site model does not change these values appreciably. The observed binding constants do not depend upon the presence or absence of the messenger RNA, poly(U). However, spectroscopic evidence strongly suggests that the anticodons of both bound tRNAs are in contact with the message. This evidence further suggests that in the presence of poly(U) the environment of the hypermodified base adjacent to the anticodon is substantially different in the two sites. This may reflect a difference in the conformation of the anticodon loops or an interaction between the hypermodified base of the weak site tRNA and the anticodon loop of the strong site tRNA.     

Discrimination between purine and pyrimidine base at the 3' terminus of the tRNA molecule by the stringent factor system from Escherichia coli.

Crude stringent factor, prepared from a mutant strain with low levels of tRNA nucleotidyl transferase, synthesizes little or no (p)ppGpp in the presence of tRNA^Phe-CpC; addition of yeast tRNA nucleotidyl transferase, however, fully restores (p)ppGpp formation, indicating that the complete CCA terminus of the tRNA molecule is a prerequisite in the (p)ppGpp synthesizing reaction. When the terminal purine is replaced by a pyrimidine base as in the case of tRNA^Phe-CpCpC; or when the latter is extended by addition of AMP yielding tRNA^Phe-CpCpCpA, both modified tRNAs are low in stimulating the (p)ppGpp synthesizing reaction. Hence activation of the stringent factor by tRNA requires (i) the terminal purine base and (ii) the precise fitting of the CCA terminus to the acceptor site of the ribosome.     

Conformational peculiarities of rRNA inff supMet from E. coli as revealed by fluorescent methods

Conformational transitions in several individual tRNAs (tRNA _inff ^supMet , tRNA^Phe from E. coli, tRNA _inf1 ^supVal , tRNA^Ser, tRNA^Phe from yeast) have been studied under various environmental conditions. The binding isotherms studies for dyes-tRNA complexes exhibited similarities in conformational states of all tRNAs investigated at low ionic strength (0.01 M NaCl). By contrast, at high ionic strength (0.4 M NaCl or 2×10^-4 M Mg²⁺) a marked difference is found in structural features of tRNA _inff ^supMet as compared with other tRNAs used. The tRNA _inff ^supMet is the only tRNA species that does not reveal the strong type of complexes with ethidium bromide, acriflavine and acridine orange.     

Identification and codon reading properties of 5-cyanomethyl uridine,a new modified nucleoside found in the anticodon wobble position of mutant haloarchaeal isoleucine tRNAs

Most archaea and bacteria use a modified C in the anticodon wobble position of isoleucine tRNA to base pair with A but not with G of the mRNA. This allows the tRNA to read the isoleucine codon AUA without also reading the methionine codon AUG. To understand why a modified C, and not U or modified U, is used to base pair with A, we mutated the C34 in the anticodon of Haloarcula marismortui isoleucine tRNA (tRNA₂^Ile) to U, expressed the mutant tRNA in Haloferax volcanii, and purified and analyzed the tRNA. Ribosome binding experiments show that although the wild-type tRNA₂^Ile binds exclusively to the isoleucine codon AUA, the mutant tRNA binds not only to AUA but also to AUU, another isoleucine codon, and to AUG, a methionine codon. The G34 to U mutant in the anticodon of another H. marismortui isoleucine tRNA species showed similar codon binding properties. Binding of the mutant tRNA to AUG could lead to misreading of the AUG codon and insertion of isoleucine in place of methionine. This result would explain why most archaea and bacteria do not normally use U or a modified U in the anticodon wobble position of isoleucine tRNA for reading the codon AUA. Biochemical and mass spectrometric analyses of the mutant tRNAs have led to the discovery of a new modified nucleoside, 5-cyanomethyl U in the anticodon wobble position of the mutant tRNAs. 5-Cyanomethyl U is present in total tRNAs from euryarchaea but not in crenarchaea, eubacteria, or eukaryotes.     

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.     

Puromycin binding to the donor (P-) site of Escherichia coli ribosomes

Puromycin inhibits the interaction of peptidyl-tRNA analogues AcPhe-tRNA_ox-red^Phe, AcPhe-tRNA^Phe and fMet-tRNA_f^Met with the donor (P-) site of Escherichia coli ribosomes. affects almost equally both the rate of the binding and the equilibrium of the system. This means that the effect is due to direct competition for the P-site, but not due to the indirect influence via the acceptor (A-) site. The inhibition was observed also in 30 S ribosomal subunits, therefore the puromycin binding site is situated far from the peptidyl transferase center. Quantitative measurements show that the affinity of puromycin for its new ribosomal binding site is similar to its affinity for the acceptor site of the peptidyl transferase center.     

A study of the interaction of Escherichia coli elongation factor-Tu with aminoacyl-tRNAs by partial digestion with cobra venom ribonuclease

The hydrolysis of several aminoacylated transfer RNAs, by double-strand-specific ribonuclease from Naja oxiana was studied. The sensitivity to this enzyme of Phe-tRNA^Phe, Glu-tRNA^Glu and Met-tRNA_m^Met from Escherichia coli and Phe-tRNA^Phe from yeast was examined, both in the free state and complexed to E. coli elongation factor Tu. The hydrolysis patterns in the isolated state were similar for all aminoacylated tRNAs except Glu-tRNA₂^Glu, which exhibited striking differences probably arising from the existence of several subpopulations of tRNA₂^Glu. When engaged in a ternary complex with EF-Tu and GTP, the aminoacyl-tRNAs were efficiently protected in the amino acid acceptor and TΨC helices, showing that the interaction with EF-Tu primarily takes place at the -C-C-A end and at the amino acid acceptor and TΨC helices. In all cases an increased reactivity of the anticodon stem was observed in the complexed tRNA, possibly resulting from a conformational change in this region of the tRNAs.     

Complex formation between transfer RNAs with complementary anticodons: use of matrix bound tRNA

It is shown that yeast tRNA^Phe, chemically coupled by its oxidized 3′CpCpA end behaves exactly as free tRNA^Phe in its ability to form a specific complex with $\text{E. coli}$ tRNA₂^Glu having a complementary anticodon. The results support models of tRNA in which the 3′CpCpA_OH end and the anticodon are not closely associated in the tertiary structure, and provide a convenient tool of general use to characterize others pairs of tRNA having complementary anticodons, as well as for highly selective purification of certain tRNA species.     

Terminal oxidation-reduction of yeast phenylalanine tRNA prevents donor and acceptor function at the peptidyl transferase center

Periodate oxidation of the ribose of the 3′-terminal adenosine of yeast tRNA^Phe followed by borohydride reduction has the net effect of splitting the C2′C3′ bond leaving two primary alcohol groups at these carbon atoms. This modified tRNA (tRNA^ox-red) could be acylated with phenylalanine but could not function as either a donor or acceptor at the peptidyl transferase center of the ribosome. Assays were performed with the phenylalanyl-pentanucleotides, CACCA^ox-red(acetylPhe) and CACCA^ox-red(Phe), which were isolated from the 3′-end of appropriately esterified tRNA^ox-red. Ado^ox-red(Phe) isolated from Phe-tRNA^ox-red was also inactive as an acceptor, but synthetic Ado^ox-red(Phe), a mixture of the 2′ and 3′ phenylalanyl esters, was active with an apparent K_m of 1.16 mM compared to 0.2 mM for control Ado(Phe). These results are interpreted to mean that (1) biosynthetic aminoacylation of tRNA^ox-red occurs specifically at the 2′-hydroxyl, (2) there is no 2′:3′ tautomerization in the ring-opened structure, and (3) peptidyl transferase recognizes specifically the 3′-aminoacyl esters of tRNA.