Effect of ribosome binding and translocation on the anticodon of tRNAPhe as studied by wybutine fluorescence.

The complexes of N-AcPhe-tRNAPhe (or non-aminoacylated tRNAPhe) from yeast with 70S ribosomes from E. coli have been studied fluorimetrically utilizing wybutine, the fluorophore naturally occurring next to the 3' side of the anticodon, as a probe for conformational changes of the anticodon loop. The fluorescence parameters are very similar for tRNA bound to both ribosomal sites, thus excluding an appreciable conformational change of the anticodon loop upon translocation. The spectral change observed upon binding of tRNAPhe to the P site even in the absence of poly(U) is similar to the one brought about by binding of poly(U) alone to the tRNA. This effect may be due to a hydrophobic binding site of the anticodon loop or to a conformational change of the loop induced by binding interactions of various tRNA sites including the anticodon.     

Stability of the unique anticodon loop conformation of E.coli tRNAfMet.

Initiator tRNAs have an anticodon loop conformation distinct from that of elongation tRNAs as detected by susceptibility to S1 nuclease. We now find the anticodon loop conformation of E. coli tRNAfMet to be stable under different salt conditions as detected by using S1 nuclease as a structural probe. In contrast, a conformational change is observed in the T- and D- loop of this tRNA in the absence of added Mg2+. This change can be suppressed by spermine. Even under those conditions effecting a change in T- and D- loop conformation, the anticodon loop does not change. This suggests that the conformational shift is controlled by Mg2+ and restricted to the D- and T- loop region only without affecting the anticodon domain. The use of S1 nuclease as a conformational probe requires the use of kinetic studies to determine the initial cleavage sites. Thus, the use of a strong inhibitor which immediately stops the action of this nuclease is necessary. ATP is shown to be such an inhibitor.     

Binding of complementary pentanucleotides to the anticodon loop of transfer RNA

The conformation of the anticodon loop of tRNA (yeast) was studied by detecting the most strongly binding pentanucleotide among the pentamers obtained by digestion of ribosomal RNA with T₁ RNase. This pentamer was identified as UUCAG which is complementary to the anticodon and the two pyrimidines on the 5′ side of the anticodon loop. Gel electrophoresis was used to detect binding. Control experiments employing other tRNA's showed that UUCAG formed a five base-pair complex with the tRNA. This indicates that the pentamer binds to the anticodon and the two pyrimidines to the 5′ side of it and lends support to a model for the tRNA loop which was recently proposed by Woese (1970).     

Nuclear magnetic resonance studies of codon-anticodon interaction in tRNAPhe. I. Effect of binding complementary tetra and pentanucleotides to the anticodon

The effect of codon-anticodon interaction on the structure of two tRNA^Phe species was investigated by means of nuclear magnetic resonance spectroscopy. To this end n.m.r.² spectra of yeast and Escherichia coli tRNA^Phe were recorded in the absence and the presence of the oligonucleotides U-U-C-A, U-U-C-G and U-U-C-A-G, which all contain the sequence UUC complementary to the anticodon sequence GAA. The spectra of the hydrogen-bonded protons, the methyl protons and the internucleotide phosphorous nuclei served to monitor the structure of the anticodon loop and of the tRNA in the tRNA-oligonucleotide complex. From the changes in the methyl proton spectra and in the phosphorous spectra it could be concluded that the oligonucleotides bind to the anticodon. Moreover it turned out that the binding constants obtained from these n.m.r. experiments were, within experimental error, equal to the values obtained with other techniques. Using the resonances of the protons hydrogen-bonded between the oligonucleotide and the anticodon loop the structure of the latter could be studied. In particular, binding of the pentanucleotide U-U-C-A-G, which is complementary to the five bases on the 5′ side of the anticodon loop, resulted in the resolution of four to five extra proton resonances indicating that four to five base-pairs are formed between the pentanucleotide and the anticodon loop. The formation of five base-pairs was confirmed by an independent fluorescence binding study. The resonance positions of the hydrogen-bonded protons indicate, that an RNA double helix is formed by the anticodon loop and U-U-C-A-G with the five base-pairs forming a continuous stack. This structure can be accomodated in the so-called 5′ stacked conformation of the anticodon loop, a structure that has been suggested earlier as an alternative to the familiar 3′ stacked conformation in the crystal structure models of yeast tRNA^Phe. It turned out that structural adjustments of the anticodon loop to the binding of the oligonucleotides are propagated into the anticodon stem. The relevance of these results with respect to the mechanism of protein synthesis is discussed.     

Codon-induced association of the isolated anticodon loop of tRNAPhe

The binding of the codon UUC to the isolated anticodon loop of tRNAPhe (yeast) has been studied as a model of codon recognition by a simple adaptor. Fluorescence titrations demonstrate that UUC binds to the isolated anticodon loop with an equilibrium constant of 1.4 X 10(3) M-1 (at 7.2 degrees C). Equilibrium sedimentation curves reveal that UUC binding induces association of anticodon loops beyond the dimer stage. A set of complete sedimentation curves obtained for various reactant concentrations was analyzed according to a model with an infinite number of subsequent association steps for UUC-anticodon loop complexes and with equal affinity for each step. The coupling of association and sedimentation was considered quantitatively, and the information resulting from conservation of mass was used by integration. According to this procedure, the experimental data can be described by an isodesmic association constant of 8 X 10(3) M-1 with satisfactory accuracy. Temperature-jump relaxation detected by fluorescence measurements provides independent evidence for codon-induced association of the anticodon loop. The data are consistent with the following mechanism: UUC preferentially binds to one of two loop conformations with a rate constant of 4.5 X 10(6) M-1 s-1; the UUC-anticodon loop complex undergoes association with a rate constant of 6.5 X 10(6) M-1 s-1. The reactions observed for the isolated anticodon loop are surprisingly similar to those observed previously for the complete tRNA, suggesting that simple hairpin loops are appropriate adaptors for a translation process at an early stage of evolution; the codon-induced association of the hairpin loop should be very useful to facilitate the transfer of cognate amino acids during translation.     

Aminoacylation of anticodon loop substituted yeast tyrosine transfer RNA

A procedure for replacing residues 33-35 in the anticodon loop of yeast tRNATyr with any desired oligonucleotide has been developed. The three residues were removed by partial ribonuclease A digestion. An oligonucleotide was inserted into the gap in four steps by using RNA ligase, polynucleotide kinase, and pseT 1 polynucleotide kinase. The rate of aminoacylation of anticodon loop substituted tRNATyr by yeast tyrosyl-tRNA synthetase was found to depend upon the sequence of the oligonucleotide inserted. This suggests that the nucleotides in the anticodon loop of yeast tRNATyr are required for optimal aminoacylation. In addition, tRNATyr modified to have a phenylalanine anticodon was shown to be misacylated by yeast phenylalanyl-tRNA synthetase at a rate at least 10 times faster than unmodified tRNATyr. Thus, the anticodon is used by phenylalanyl-tRNA synthetase to distinguish between tRNAs.     

Probing the anticodon loop structure in yeast tRNA(Phe-Y) with single strand-specific nuclease S1

Yeast tRNA(Phe) and tRNA(Phe-Y) are cleaved by single strand-specific endonuclease S1 at the same positions within the anticodon loop (phosphates 34, 36 and 37) and at the 3'-terminus (phosphates 75 and 76). The efficiency of the anticodon loop hydrolysis is much higher in tRNA(Phe-Y) while the cutting at the 3'-terminus is not influenced considerably by the Y-base1 removal from yeast tRNA(Phe). The effect of the Y-base excision on the structure of the anticodon loop is discussed on the basis of the S1 digestion studies as well as other relevant results.     

The bases of the tRNA anticodon loop are independent by genetic criteria.

We employed two methods to study the translational role of interactions between anticodon loop nucleotides. Starting with a set of previously constructed weakly-suppressing anticodon loop mutants of Su7, we searched for second-site revertants that increase amber suppressor efficiency. Though hundreds of revertants were characterized, no second-site revertants were found in the anticodon loop. Second site reversion was detected in the D-stem, thereby demonstrating the efficacy of the search method. As a second method for detecting interactions, we used site-directed mutagenesis to construct multiple mutations in the anticodon loop. These multiple mutants are very weak suppressors and have translational activities that are equal to or lower than that predicted for the independent action of single mutations. We conclude that although the anticodon loop sequence of Su7 has an optimal structure for the translation of amber codons, we find no evidence that interactions between loop bases can enhance translational efficiency.     

What sequence must have an 8-nucleotide anticodon loop so that tRNA would not shift the reading frame?

A model describing the interaction between the anticodon loop consisting of eight nucleotides and messenger RNA is proposed. A suggestion is made that the ability of eight nucleotide loop to recognize a four base codon and as a result to shift a reading frame, depends on the location of the sharp turn of polynucleotide chain within the anticodon loop. The turn will appear to be only immediately after U. This U fixes the position of the turn by the formation of the specific hydrogen bond and van-der-Vaals contacts, that are found in the common seven nucleotide anticodon loop. The model predicts whether a particular tRNA will be able to shift a reading frame or not. This prediction is in agreement with available experimental data.     

Topological arrangement of two transfer RNAs on the ribosome. Fluorescence energy transfer measurements between A and P site-bound tRNAphe

The relative arrangement of two tRNAPhe molecules bound to the A and P sites of poly(U)-programmed Escherichia coli ribosomes was determined from the spatial separation of various parts of the two molecules. Intermolecular distances were calculated from the fluorescence energy transfer between fluorophores in the anticodon and D loops of yeast tRNAPhe. The energy donors were the natural fluorescent base wybutine in the anticodon loop or proflavine in both anticodon (position 37) and D loops (positions 16 and 17). The corresponding energy acceptors were proflavine or ethidium, respectively, at the same positions. Four distances were measured: anticodon loop-anticodon loop, 24(+/- 4) A; anticodon loop (A site)-D loop (P site), 46(+/- 12) A: anticodon loop (P site)-D loop (A site), 38(+/- 10) A: D loop-D loop, 35(+/- 9) A. Assuming that both tRNAs adopt the conformation present in the crystal and that the CCA ends are close to each other, the results are consistent with the two anticodons being bound to contiguous codons and suggest an asymmetric arrangement in which the planes of the two L-shaped molecules enclose an angle of 60 degrees +/- 30 degrees.     

Involvement of the size and sequence of the anticodon loop in tRNA recognition by mammalian and E. coli methionyl-tRNA synthetases.

The rates of the cross-aminoacylation reactions of tRNAs(Met) catalyzed by methionyl-tRNA synthetases from various organisms suggest the occurrence of two types of tRNA(Met)/methionyl-tRNA synthetase systems. In this study, the tRNA determinants recognized by mammalian or E. coli methionyl-tRNA synthetases, which are representative members of the two types, have been examined. Like its prokaryotic counterpart, the mammalian enzyme utilizes the anticodon of tRNA as main recognition element. However, the mammalian cytoplasmic elongator tRNA(Met) species is not recognized by the bacterial synthetase, and both the initiator and elongator E. coli tRNA(Met) behave as poor substrates of the mammalian cytoplasmic synthetase. Synthetic genes encoding variants of tRNAs(Met), including the elongator one from mammals, were expressed in E. coli. tRNAs(Met) recognized by a synthetase of a given type can be converted into a substrate of an enzyme of the other type by introducing one-base substitutions in the anticodon loop or stem. In particular, a reduction of the size of the anticodon loop of cytoplasmic mammalian elongator tRNA(Met) from 9 to 7 bases, through the creation of an additional Watson-Crick pair at the bottom of the anticodon stem, makes it a substrate of the prokaryotic enzyme and decreases its ability to be methionylated by the mammalian enzyme. Moreover, enlarging the size of the anticodon loop of E. coli tRNA(Metm) from 7 to 9 bases, by disrupting the base pair at the bottom of the anticodon stem, renders the resulting tRNA a good substrate of the mammalian enzyme, while strongly altering its reaction with the prokaryotic synthetase. Finally, E. coli tRNA(Metf) can be rendered a better substrate of the mammalian enzyme by changing its U33 into a C. This modification makes the sequence of the anticodon loop of tRNA(Metf) identical to that of cytoplasmic initiator tRNA(Met).     

Enzymatic alteration of the anticodon IAC of Torulopsis tRNAVal to IAU for isoleucine anticodon and aminoacylation of the variant

By enzymatic cleavage and ligation of tRNAVa1, its anticodon sequence IAC was altered to IAU, the anticodon of tRNAI1e. Valine acceptor activity of this variant tRNAVa1 (IAU) was reduced to the extent much lower than tyrosine acceptability of the previously prepared tRNATyr (GAA) (anticodon for tRNAPhe). Isoleucine acceptor activity was undetected, contrary to tRNATyr (GAA) which accepted phenylalanine weakly. Cleavage of tRNAVa1 (IAC) between IACA37 and C38 of its anticodon loop reduced the valine acceptor activity, suggesting some contribution of the conformation of the anticodon loop to the aminoacylation reaction.     

Starvation-induced cleavage of the tRNA anticodon loop in Tetrahymena thermophila

Amino acid deprivation triggers dramatic physiological responses in all organisms, altering both the synthesis and destruction of RNA and protein. Here we describe, using the ciliate Tetrahymena thermophila, a previously unidentified response to amino acid deprivation in which mature transfer RNA (tRNA) is cleaved in the anticodon loop. We observed that anticodon loop cleavage affects a small fraction of most or all tRNA sequences. Accumulation of cleaved tRNA is temporally coordinated with the morphological and metabolic changes of adaptation to starvation. The starvation-induced endonucleolytic cleavage activity targets tRNAs that have undergone maturation by 5' and 3' end processing and base modification. Curiously, the majority of cleaved tRNAs lack the 3' terminal CCA nucleotides required for aminoacylation. Starvation-induced tRNA cleavage is inhibited in the presence of essential amino acids, independent of the persistence of other starvation-induced responses. Our findings suggest that anticodon loop cleavage may reduce the accumulation of uncharged tRNAs as part of a specific response induced by amino acid starvation.     

Anticodon loop mutations perturb reading frame maintenance by the E site tRNA

The ribosomal E site helps hold the reading frame. Certain tRNA mutations affect translation, and anticodon loop mutations can be especially detrimental. We studied the effects of mutations saturating the anticodon loop of the amber suppressor tRNA, Su7, on the ability to help hold the reading frame when in the E site. We also tested three mutations in the anticodon stem, as well as a mutation in the D stem (the “Hirsh” mutation). We used the Escherichia coli RF2 programmed frameshift site to monitor frame maintenance. Most anticodon loop mutations increase frameshifting, possibly by decreasing codon:anticodon stability. However, it is likely that the A site is more sensitive to anticodon loop structure than is the E site. Unexpectedly, the Hirsh mutation also increases frameshifting from the E site. Other work shows that mutation may increase the ability of tRNA to react in the A site, possibly by facilitating conformational changes required for aminoacyl-tRNA selection. We suggest that this property may decrease its ability to bind to the E site. Finally, the absence of the ms²io⁶A nucleoside modifications at A37 does not decrease the ability of tRNA to help hold the reading frame from the E site. This was also unexpected because the absence of these modifications affects translational properties of tRNA in A and P sites. The absence of a negative effect in the E site further highlights the differences among the substrate requirements of the ribosomal coding sites.     

Analysis of magnesium, europium and lead binding sites in methionine initiator and elongator tRNAs by specific metal-ion-induced cleavages

The specificity of cleavages in yeast and lupin initiator and elongator methionine tRNAs induced by magnesium, europium and lead has been analysed and compared with known patterns of yeast tRNA(Phe) hydrolysis. The strong D-loop cleavages occur in methionine elongator tRNAs at similar positions and with comparable efficiency to those found in tRNA(Phe), while the sites of weak anticodon loop cuts, identical in methionine elongator tRNAs, differ from those found in tRNA(Phe). Methionine initiator tRNAs differ from their elongator counterparts: (a) they are cleaved in the D-loop with much lower efficiency; (b) they are cleaved in the variable loop which is completely resistant to hydrolysis in elongator tRNAs; (c) cleavages in the anticodon loop are stronger in initiator tRNAs and they are located mostly at the 5' side of the loop whereas in elongator tRNAs they occur mostly at the opposite, 3' side of the loop. The distinct pattern of the anticodon loop cleavages is considered to be related to different conformations of the anticodon loop in the two types of methionine tRNAs.     

Role of the constant uridine in binding of yeast tRNAPhe anticodon arm to 30S ribosomes.

Twenty-two anticodon arm analogues were prepared by joining different tetra, penta, and hexaribonucleotides to a nine nucleotide fragment of yeast tRNAPhe with T4 RNA ligase. The oligomer with the same sequence as the anticodon arm of tRNAPhe bind poly U programmed 30S ribosomes with affinity similar to intact tRNAPhe. Analogues with an additional nucleotide in the loop bind ribosomes with a weaker affinity whereas analogues with one less nucleotide in the loop do not bind ribosomes at all. Reasonably tight binding of anticodon arms with different nucleotides on the 5' side of the anticodon suggest that positions 32 and 33 in the tRNAPhe sequence are not essential for ribosome binding. However, differences in the binding constants for anticodon arms containing modified uridine residues in the "constant uridine" position suggest that both of the internal "U turn" hydrogen bonds predicted by the X-ray crystal structure are necessary for maximal ribosome binding.     

Secondary structure in formylmethionine tRNA influences the site-directed cleavage of ribonuclease H using chimeric 2'-O-methyl oligodeoxyribonucleotides

In order to cleave RNA at specific positions in Escherichia coli formylmethionine tRNA, RNase H and complementary chimeric oligonucleotides consisting of DNA and 2'-O-methyl-RNA (Inoue et al. (1987) FEBS Lett. 215, 327] were used. Specific cleavages in the D loop, anticodon loop, T psi C loop, anticodon stem, and acceptor stem were investigated. Virtually unique hydrolyses with RNase H were observed at the T psi C loop, anticodon stem, and acceptor stem when relatively longer chimeric oligonucleotides (20-mer) were used. An efficient cleavage at the anticodon was obtained with a chimeric 13-mer when the higher structure of the tRNA was broken by hybridization with a 20-mer at the acceptor as well as the T psi C stem region. It was found that stabilities of hybrids with chimeric oligonucleotides and the presence of minor nucleosides affect the cleavage of tRNA by this approach.     

Effect of translocation on topology and conformation of anticodon and D loops of tRNAPhe

Steady-state fluorescence and fluorescence anisotropy measurements have been carried out on isolated complexes of fluorescent derivatives of N-AcPhe-tRNA^Phe with 70 S ribosomes from Escherichia coli. As a fluorescent probe, proflavine was inserted into either the anticodon loop or the D loop.Upon binding to the A site of poly(U)-programmed ribosomes, the probe in the anticodon loop is highly immobilized and effectively shielded against solvent access in a hydrophobic binding site. Elongation factor G-dependent translocation to the P site does not change any of the fluorescence parameters. These observations indicate that in both sites the environment of the probe with respect to hydrophobicity and shielding against solvent access is rather similar. Moreover, substantial conformational changes of the anticodon loop upon translocation are made unlikely.In contrast to the anticodon loop, the D loop is fully exposed to the solvent in both A and P sites, indicating that the variable region in the middle of the D loop is oriented away from the ribosomal surface.On the other hand, depolarization measurements show that the D loop is strongly immobilized in the A site, possibly by binding interactions of invariant bases of the loop. Upon translocation, the D loop gains considerable flexibility, indicating that in the P site it is neither fixed by contacts with the ribosome nor by intramolecular base-pairing with the T loop.In the absence of poly(U) or in the presence of poly(C), the fluorescence parameters of the probes in the anticodon loop and, more significantly, in the D loop, differ from those observed in the presence of poly(U). These differences are best explained by assuming a codon-induced conformational change of the anticodon loop, which in turn is transmitted to the D loop.When the non-aminoacylated tRNA^Phe derivatives are studied, spectroscopic differences as compared to the respective N-AcPhe-tRNA^Phe derivatives are observed only for the A site complexes. It appears that the aminoacylation influences the binding of transfer RNA in the A site, but not in the P site.     

Enzymatic 2''-O-methylation of the wobble nucleoside of eukaryotic tRNAPhe: specificity depends on structural elements outside the anticodon loop.

We have investigated the specificity of the enzyme tRNA (wobble guanosine 2'-O-)methyltransferase which catalyses the maturation of guanosine-34 of eukaryotic tRNAPhe to the 2'-O-methyl derivative Gm-34. This study was done by micro-injection into Xenopus laevis oocytes of restructured yeast tRNAPhe in which the anticodon GmAA and the 3' adjacent nucleotide 'Y' were substituted by various tetranucleotides. The results indicate that the enzyme is cytoplasmic; the chemical nature of the bases of the anticodon and its 3' adjacent nucleotide is not critical for the methylation of G-34; the size of the anticodon loop is however important; structural features beyond the anticodon loop are involved in the specific recognition of the tRNA by the enzyme since Escherichia coli tRNAPhe and four chimeric yeast tRNAs carrying the GAA anticodon are not substrates; unexpectedly, the 2'-O-methylation is not restricted to G-34 since C-34, U-34 and A-34 in restructured yeast tRNAPhe also became methylated. It seems probable that the tRNA (wobble guanosine 2'-O-)methyltransferase is not specific for the type of nucleotide-34 in eukaryotic tRNAPhe; however the existence in the oocyte of several methylation enzymes specific for each nucleotide-34 has not yet been ruled out.