tRNA topography during translocation: steady-state and kinetic fluorescence energy-transfer studies

The distances between the anticodon loops of fluorescent tRNAPhe bound to the E site and to either the A or the P site of poly(U)-programmed Escherichia coli ribosomes were measured by fluorescence energy transfer. Donor and acceptor molecules were wybutine and proflavin, respectively, both located 3' to the anticodon of tRNAPhe. The anticodon loops were found to be separated by 42 +/- 10 A (A to E site) and 34 +/- 8 A (P to E site). The latter distance is much larger than the one measured between the anticodon loops of A and P site bound tRNAs [24 +/- 4 A; Paulsen, H., Robertson, J. M., & Wintermeyer, W. (1983) J. Mol. Biol. 167, 411-426], rendering unlikely simultaneous codon-anticodon interaction in the P and E sites. In kinetic stopped-flow measurements, the energy transfer between the anticodon loops of the tRNA molecules was followed during translocation. The transfer efficiency decreases in three steps with apparent rate constants on the order of 1, 0.1, and 0.01 s-1. The fast step is ascribed to the simultaneous displacement of the deacylated tRNAPhe out of the P site and of the N-AcPhe-tRNAPhe from the A site to the P site. The distance between the anticodon loops does not change appreciably during this reaction. A significant separation of the two tRNAs occurs during the intermediate and the slow steps. The latter most likely represents a rearrangement of the posttranslocation complex containing both tRNA molecules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pre-steady-state kinetics of ribosomal translocation

The two partial reactions of elongation factor G dependent translocation, the release of deacylated tRNA from the P site and the displacement of peptidyl tRNA from the A to the P site, have been studied with the stopped-flow technique. The experiments were performed with poly(U)-programmed ribosomes from Escherichia coli carrying deacylated tRNAPhe in the P site and N-AcPhe-tRNAPhe in the A site in the presence of GTP. The kinetics of the reaction were followed by monitoring either the intensity or the polarization of the fluorescence of both wybutine and proflavine located in the anticodon loop or of proflavine located in the D loop of yeast tRNAPhe or N-AcPhe-tRNAPhe. Both displacement and release fluorescence changes could be described by three exponentials, exhibiting apparent first-order rate-constants (20 degrees C) of 2 to 5 s-1 (15 s-1, 35 degrees C), 0.1 to 0.3 s-1, and 0.01 to 0.02 s-1, measured with a saturating concentration of elongation factor G (1 microM). The activation energy for the fast process of both reactions was found to be 70 kJ/mol (17 kcal/mol), while the intermediate process exhibits an activation energy of 30 kJ/mol (7 kcal/mol). The fast step is assigned to the displacement of the N-AcPhe-tRNAPhe from the A to the P site, and to the release of the tRNAPhe from the P site. The reactions take place simultaneously to form an intermediate post-translocation complex. The latter, in the intermediate step, rearranges to form a post-translocation complex carrying the deacylated tRNAPhe in an exit site and N-AcPhe-tRNAPhe in the P site, both in their equilibrium states. In parallel, or subsequently, the deacylated tRNAPhe spontaneously dissociates from the ribosome, thus completing the translocation process. The slow process has not been assigned.     

Comparison of ribosomal entry and acceptor transfer ribonucleic acid binding sites on Escherichia coli 70S ribosomes. Fluorescence energy transfer measurements from Phe-tRNAPhe to the 3' end of 16S ribonucleic acid

Distances were measured by nonradiative energy transfer from fluorescent probes specifically located on one of three points of yeast or Escherichia coli Phe-tRNAPhe enzymatically bound to the entry site or to the acceptor site of E. coli 70S ribosomes to energy-accepting probes on the 3' end of the 16S ribonucleic acid (RNA) of the 30S subunit. The Y base in the anticodon loop of yeast tRNAPhe was replaced by proflavin. Fluorescein isothiocyanate was attached to the X base (position 47) of E. coli tRNAPhe. E. coli tRNAPhe which had been photochemically cross-linked between positions 8 and 13 followed by chemical reduction to form a fluorescent probe was also used. Labeled tRNAs were aminoacylated and enzymatically bound to the ribosome in the presence of elongation factor Tu and guanosine 5'-triphosphate (acceptor-site binding) or a nonhydrolyzable analogue (entry-site binding). Nonradiative energy transfer measurements were made of the distances between fluorophores located on the Phe-tRNA and the fluorophore at the 3' end of 16S RNA. Calculations were based on comparison of the fluorescence lifetime of the energy donor, located on the Phe-tRNA, in the absence and presence of an energy acceptor on the 3' end of the 16S RNA. Under both sets of binding conditions, the distances to the 3' end of 16S RNA were found to be the following: cross-linked tRNA, greater than 69 A; Y base of tRNA, greater than 61 A. The distance between the 3' end of 16S RNA and the X base of tRNA was found to be 81 A under acceptor-site binding conditions but greater than 86 A under entry-site binding conditions.     

Fluorescence decay and quantum yield characteristics of acridine orange and proflavine bound to DNA

Fluorescence properties (quantum yield, decay curve, lifetime and polarization) of acridine orange and proflavine bound to DNA were examined as a function of nucleotide to dye (P/D) ratio. First, mean fluoiescence lifetimes were determined by the phase-shift measurements. The lifetime and quantum yield of acridine orange increased in a parallel fashion with increasing P/D ratio. There was no parallel relation between the lifetime and quantum yield for proflavine; the lifetime showed a minimum around P/D = 10. Next, fluorescence decay curves were measured by the monophoton counting technique and analyzed with the aid of the method of moments and the Laplace transform method. The results showed that the fluorescence decay of bound acridine orange was exponential above P/D = 10. On the other hand, the decay of bound proflavine was exponential above P/D = 100, but markedly deviated from exponentiality with decreasing P/D ratio. The results of fluorescence polarization suggested that this phenomenon is the result of Förster energy transfer between proflavine molecules bound to the fluorescent site (AT pair) and bound to the quenching site (GC pair). Critical transfer distances were 26-4 and 37.0 Å, respectively, for bound proflavine and acridine orange.     

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.     

Poly(U)-dependent interaction of yeast tRNA(Phe) and its fragments with ribosomes from Escherichia coli

The poly(U)-dependent bindings of yeast tRNAPhe, its derivative depleted of 3'-terminal adenosine, and 15-nucleotide having a sequence of yeast tRNAPhe anticodon arm to the P site of Escherichia coli 70S ribosomes were compared. The equilibrium and rate constants were determined. Data indicate that the anticodon arm (N28-N42) contributes the major fraction of the binding free energy (-45.3 kJ/mol at 10 mM Mg2+ and 30 degrees C). Other parts of the tRNAPhe molecule besides A76 (N1-N27 and N43-N75) bring additional-6.0 kJ/mol, and A76 contributes-2.4 kJ/mol.     

A novel conformational change of the anticodon region of tRNAPhe (yeast).

The temperature dependence of the fluorescence of the Y-base of tRNAPhe (yeast) was investigated kinetically by the temperature jump method. In the range between -15 degrees C and +30 degrees C A NOVEL CONFORMATIONAL TRANSITION OF THE TRNA could be characterized. This conformational change was found in the absence of any artificial label; it is a characteristic property of tRNAPhe in its native structure. This transition accounts for 30% of the total fluorescence change. Its activation enthalpy is 16 kcal/mole (67 kJ/mole), and the transition enthalpy is between -2 kcal/mole and +2 kcal/mole (+/-8 kJ/mole). A model is represented in which this transition can be explained by a a change in the stacking pattern of the anticodon loop. The experimental findings are discussed with respect to several hypotheses about the molecular mechanism of protein biosynthesis which postulate conformational rearrangements of the anticodon loop.     

High-resolution phosphorus nuclear magnetic resonance spectroscopy of transfer ribonucleic acids: multiple conformations in the anticodon loop

The temperature dependence of the 31P NMR spectra of yeast phenylalanine tRNA, E. coli tyrosine, glutamate (2), and formylmethionine tRNA is presented. The major difference between the 31P NMR spectra of the different acceptor tRNAs is in the main cluster region between -0.5 and -1.3 ppm. This confirms an earlier assignment of the main cluster region to the undistorted phosphate diesters in the hairpin loops and helical stems. In addition the 31P NMR spectra for all tRNAs reveal approximately 16 nonhelical diester signals spread over approximately 7 ppm besides the downfield terminal 3'-phosphate monoester. In the presence of 10 mM Mg2+ most scattered and main cluster signals do not shift between 22 and 66 degrees C, thus supporting our earlier hypothesis that 31P chemical shifts are sensitive to phosphate ester torsional and bond angles. At greater than 70 degrees C, all of the signals merge into a single random-coil conformation signal. A number of the scattered peaks are shifted (0.2-1.7 ppm) and broadened between 22 and 66 degrees C in the presence of Mg2+ and spermine as a result of a conformational transition in the anticodon loop. The 31P NMR spectrum of the dimer formed between yeast tRNAPhe and E. coli tRNA 2Glu is reported. This dimer simulates codon-anticodon interaction since the anticodon triplets of the two tRNAs are complementary. Evidence is presented that the anticodon-anticodon interaction alters the anticodon conformation and partially disrupts the tertiary structure of the tRNA.     

Anticodon loop of tRNAPhe: structure, dynamics, and Mg2+ binding

The structure, dynamics, and Mg2+ binding reactions of the isolated anticodon hairpin loop from tRNAPhe (yeast) have been analyzed by fluorescence-detected temperature-jump relaxation, melting experiments, and equilibrium sedimentation. Most of the measurements were performed at an ionic strength of 0.15 M and at temperatures below 25 degrees C, where the hairpin loop proved to be stable. A relaxation effect with a time constant of approximately 100 microseconds, indicated by the Wye base fluorescence, is attributed to a conformational change of the anticodon loop and is very similar to a corresponding transition observed previously for the whole tRNAPhe molecule. A Mg2+ binding site reflected by an inner-sphere relaxation process and associated with a strong increase of the Wye base fluorescence closely resembles a corresponding site observed in the complete tRNAPhe and is attributed to a site in the anticodon loop identified by X-ray analysis. In addition to the Mg2+ site in the loop, which is associated with a binding constant of 2 X 10(3) M-1, the existence of sites with a higher affinity is demonstrated by an unusual relaxation effect, showing a minimum in the reciprocal time constant with increasing Mg2+ concentration. The experimental data can be described by a transition between two states and Mg2+ binding to both states resulting in a reaction cycle, which is extended by an additional Mg2+ binding reaction to one of the states. The unusual effect has not been observed for the complete tRNAPhe and is also not observed when Ca2+ is added instead of Mg2+. This result indicates the existence of a conformational change involving Mg2+ inner-sphere complexation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of Escherichia coli tRNAPhe in the free state, in the ternary complex and in the ribosomal A and P sites by chemical probing

tRNAPheE.coli was modified at accessible guanosine, cytidine, and adenosine residues using the chemical modification method described by Peattie and Gilbert [Proc. Natl Acad. Sci. USA, 77, 4679-4689 (1980)]. Modification characteristics of the tRNA in the free state, in the ternary complex with elongation factor EF-Tu and GTP and in the ribosomal A and P sites were compared. A special procedure was devised to monitor, exclusively, tRNA molecules in the aminoacylated state. In the free tRNA, the most reactive bases are confined to the A73-C-C-A sequence of the aminoacyl stem, the anticodon loop, the D-loop and the extra loop and the results correlate well with the three-dimensional structure of tRNAPheyeast determined by X-ray studies. The pattern of reactivity was not affected either by charging the tRNA with phenylalanine or by labelling the 3' terminus with pCp. In the ternary complex, with elongation factor EF-Tu and GTP, changes in modification were observed at two sites, A73-C-C-A at the 3' terminus and C-13 and C-17 in the D-loop region, which are about 6 nm apart; no difference was observed in the anticodon loop. tRNAPhe bound at the ribosomal A or P sites exhibited similar, but not identical, modification patterns. Whereas nucleotides C-74 and C-75 were strongly protected at both sites, the adjacent A-73 showed an enhanced reactivity in the A site. The anticodon region G34-A-A-ms2.6(1)A was also strongly protected at both sites. In addition, nucleotide A-21 was protected during A-site, but not P-site, binding.     

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.     

The solution structure of a RNA pentadecamer comprising the anticodon loop and stem of yeast tRNAPhe. A 500 MHz 1H-n.m.r. study.

A 500 MHz 1H-n.m.r. study on the semi-synthetic RNA pentadecamer 5'-r(C-A-G-A-Cm-U-Gm-A-A-Y-A-psi-m5C-U-G) comprising the anticodon loop and stem (residues 28-42) of yeast tRNAPhe is presented. By using pre-steady-state nuclear-Overhauser-effect measurements all exchangeable and non-exchangeable base proton resonances, all H1' ribose resonances and all methyl proton resonances are assigned and over 70 intra- and inter-nucleotide interproton distances determined. From the distance data the solution structure of the pentadecamer is solved by model-building. It is shown that the pentadecamer adopts a hairpin-loop structure in solution with the loop in a 3'-stacked conformation. This structure is both qualitatively and quantitatively remarkably similar to that of the anticodon loop and stem found in the crystal structures of tRNAPhe with an overall root-mean-square difference of 0.12 nm between the interproton distances determined by n.m.r. and X-ray crystallography. The hairpin-loop solution structure of the pentadecamer is very stable with a 'melting' temperature of 53 degrees C in 500 mM-KCl, and the structural features responsible for this high stability are discussed. Interaction of the pentadecamer with the ribotrinucleoside diphosphate UpUpC, one of the codons for the amino acid phenylalanine, results only in minor perturbations in the structure of the pentadecamer, and the 3'-stacked conformation of the loop is preserved. The stability of the pentadecamer-UpUpC complex (K approximately 2.5 X 10(4) M-1 at 0 degrees C) is approximately an order of magnitude greater than that of the tRNAPhe-UpUpC complex.     

Theoretical study of hydration of RNA.

The hydration phenomena of A-RNA double helix and the anticodon loop of transfer RNA have been theoretically investigated using the empirical potential energy functions. The hydration schemes of a model compound of A-RNA and a polynucleotide which has the structure of the anticodon loop of yeast tRNAPhe have been determined, and their stabilization energies produced by the introduction of water in the first hydration shell were calculated by considering the hydrated ones as supermolecules. The results indicate that hydration scheme of A-RNA considerably differs from that of B-DNA and stabilization energy due to hydration of A-RNA is not so great as B-DNA. In the anticodon loop structure, however, stabilizing effect of the bound water molecules upon the structure is significant. From the results, the reason why the structure of RNA remains unchanged with the change of hydration degree while that of DNA is altered was studied.     

The solution structure of the Escherichia coli initiator tRNA and its interactions with initiation factor 2 and the ribosomal 30 S subunit

The conformation of the Escherichia coli initiator tRNA has been investigated using enzymatic and chemical probes. This study was conducted on the naked tRNA and on the tRNA involved in the various steps leading to the formation of the 30 S.IF-2.GTP.fMet-tRNA.AUG complex. A three-dimensional model of the initiator tRNA is presented, which displays several differences with yeast tRNAPhe: (i) the anticodon arm is more rigid; (ii) the presence of an additional nucleotide in the D loop results in specific features in both T and D loops; (iii) C1 and A72 might form a noncanonical base pair. Aminoacylation and formylation induce subtle conformational adjustments near the 3' end, the T arm and the D loop. Initiation factor (IF) 2 interacts with a rather limited portion of the tRNA, covering the T loop and the minor groove of the T stem, and induces an increased flexibility in the anticodon arm. The specific structural features observed in the T loop are probably recognized by IF-2. In the 30 S.IF-2.GTP.fMet-tRNA.AUG complex, additional protections are observed in the acceptor stem and in the anticodon arm, resulting from a strong steric hindrance and from the codon-anticodon interaction within the subunit decoding site.     

Fluorescent derivatives of yeast tRNAPhe.

The preparation of four fluorescent derivatives of tRNAPhe (yeast) and their characterization by chemical, spectroscopic, and biochemical methods is described. The derivatives are prepared by replacing wybutine (position 37 in the anticodon loop) or NaBH4-reduced dihydrouracil (positions 16/17 in the hU loop) with ethidium or proflavine; they are isolated by reversed-phase chromatography (RPC-5). All tRNAPhe-dye derivatives are aminoacylated by yeast phenylalanyl-tRNA synthetase to at least 80% of the charging capacity of the unmodified tRNAPhe with an unchanged Km (0.2 mucroM) and a V lowered by 30--50%. They exhibit good to excellent activity in the aminoacylation assay from synthetase from Escherichia coli. It is concluded that the insertion of the dyes does not seriously disturb essential elements of the native tRNAPhe structure. The dyes are bound via N-ribosylic linkages. The appearance of isomeric tRNAPhe-ethidium derivatives is attributed to the involvement of the different amino groups of ethidium in the condensation. In addition, there are indications for the existence of alpha and beta anomers of the tRNA-dye compounds. The dyes are rigidly fixed to their position in the tRNA molecule by stacking interactions with the neighboring bases. The ethidium probes show Mg2+-induced changes of the tRNA conformation which are paralleled by changes of the rate of aminoacylation. On the basis of this observation it is hypothesized that conformational flexibility of the tRNA molecule is a functionally important feature of the tRNA structure.     

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.     

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.     

The structure of yeast tRNA(Asp). A model for tRNA interacting with messenger RNA

The anticodon of yeast tRNA(Asp), GUC, presents the peculiarity to be self-complementary, with a slight mismatch at the uridine position. In the orthorhombic crystal lattice, tRNA(Asp) molecules are associated by anticodon-anticodon interactions through a two-fold symmetry axis. The anticodon triplets of symmetrically related molecules are base paired and stacked in a normal helical conformation. A stacking interaction between the anticodon loops of two two-fold related tRNA molecules also exists in the orthorhombic form of yeast tRNA(Phe). In that case however the GAA anticodon cannot be base paired. Two characteristic differences can be correlated with the anticodon-anticodon association: the distribution of temperature factors as determined from the X-ray crystallographic refinements and the interaction between T and D loops. In tRNA(Asp) T and D loops present higher temperature factors than the anticodon loop, in marked contrast to the situation in tRNA(Phe). This variation is a consequence of the anticodon-anticodon base pairing which rigidifies the anticodon loop and stem. A transfer of flexibility to the corner of the tRNA molecule disrupts the G19-C56 tertiary interactions. Chemical mapping of the N3 position of cytosine 56 and analysis of self-splitting patterns of tRNA(Asp) substantiate such a correlation.     

Probing tRNA binding sites on the Escherichia coli 30 S ribosomal subunit with photoreactive analogs of the anticodon arm

Two analogs of the anticodon arm of yeast tRNAPhe (residues 28-43), in which G43 was replaced by the photoreactive nucleosides 2-azidoadenosine and 8-azidoadenosine, have been used to create 'zero-length' cross-links to ribosomal components at the peptidyl-tRNA binding site (P site) of 30 S subunits from the Escherichia coli ribosome. To prepare the analogs, 2-azidoadenosine and 8-azidoadenosine bisphosphates were first ligated to the 3' end of the anticodon-containing dodecanucleotide ACmUGmAAYA psi m5CUG from yeast tRNAPhe. The trinucleotide CAG was then joined to the 5' end of the resulting tridecanucleotide in a subsequent ligation. Both analogs bound to poly(U)-programmed 30 S subunits with affinities similar to that of the unmodified anticodon arm from yeast tRNAPhe. Irradiation of noncovalent complexes containing the photolabile analogs, poly(U) and 30 S ribosomal subunits with 300 nm light led to the covalent attachment of the anticodon arms to proteins S13 and S19. Further analysis revealed that S13 accounted for about 80%, and S19 for about 20%, of the cross-linked material. Labeling of these two proteins with 'zero-length' cross-linking probes provides useful information about the location and orientation of P site-bound tRNA on the ribosome and permits a test of recently proposed models of the three-dimensional structure of the 30 S subunit.     

Models for two tRNAs bound to successive codons on mRNA on the ribosome

We have investigated the structural changes necessary to build a model complex of two molecules of phenylalanine transfer RNA (tRNA(Phe) bound to successive codons in a short segment of a model messenger RNA (mRNA), consisting of U6. We keep the mRNA in an ideal helical conformation, deforming the tRNAs as necessary to eliminate steric overlaps while bringing the two 3' termini together. The resulting model has the two tRNAs oriented relative to one another in a manner that is very similar to a model developed by McDonald and Rein (1) in which the tRNAs maintain their ideal crystallographic conformations and all of the deformations are introduced into the mRNA. Consequently, regardless of how one divides the deformations between the tRNAs and the mRNA it is clear that, on the ribosome, the tRNA in the P site has its "front" side (that side with the variable loop) close to the "back" side of the tRNA in the A site (that side with the D loop). The space between the two molecules must be left free on the ribosome, in order to facilitate the transition from the A site to the P site. A detailed pathway is also proposed for changing the anticodon loop structure from that of the A site to that of the P site. The anticodon loop is always kept in a 3'-stacked conformation, since we find that the shift between the 3'-stacked and 5'-stacked structures proposed by Woese (2) is not feasible.