Wolfgang Wintermeyer

Binding of the 3' terminus of tRNA to 23S rRNA in the ribosomal exit site actively promotes translocation.

A key event in ribosomal protein synthesis is the translocation of deacylated tRNA, peptidyl tRNA and mRNA, which is catalyzed by elongation factor G (EF‐G) and requires GTP. To address the molecular mechanism of the reaction we have studied the functional role of a tRNA exit site (E site) for tRNA release during translocation. We show that modifications of the 3′ end of tRNAPhe, which considerably decrease the affinity of E‐site binding, lower the translocation rate up to 40‐fold. Furthermore, 3′‐end modifications lower or abolish the stimulation by P site‐bound tRNA of the GTPase activity of EF‐G on the ribosome. The results suggest that a hydrogen‐bonding interaction of the 3′‐terminal adenine of the leaving tRNA in the E site, most likely base‐pairing with 23S rRNA, is essential for the translocation reaction. Furthermore, this interaction stimulates the GTP hydrolyzing activity of EF‐G on the ribosome. We propose the following molecular model of translocation: after the binding of EF‐G.GTP, the P site‐bound tRNA, by a movement of the 3′‐terminal single‐stranded ACCA tail, establishes an interaction with 23S rRNA in the adjacent E site, thereby initiating the tRNA transfer from the P site to the E site and promoting GTP hydrolysis. The co‐operative interaction between the E site and the EF‐G binding site, which are distantly located on the 50S ribosomal subunit, is probably mediated by a conformational change of 23S rRNA.

Splitting of phenylalanine specific tRNA into half molecules by chemical means

Abstract When the base moiety of the unknown nucleoside Y of phenylalanine specific tRNA from yeast is excised, the tRNA can be split into two large fragments under the conditions of the Whitfeld degradation. The chain scission is a completely specific, almost quantitative process. The mixture of the two fragments has 50–60% of the acceptor activity for phenylalanine as compared with unmodified phenylalanine specific tRNA.

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-tRNAPhe with 70 S ribosomes from Escherichia coli. As a fluorescent probe, proflavine was inserted into either the anticodon loop or the D loop. n nUpon 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. n nIn 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. n nOn 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. n nIn 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. n nWhen the non-aminoacylated tRNAPhe derivatives are studied, spectroscopic differences as compared to the respective N-AcPhe-tRNAPhe 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.

Cooperative helix-coil transitions in half molecules of phenylalanine specific tRNA from yeast

The mechanism of helix-coil transitions in synthetic homo-oligonucleotides has been elucidated by recent kinetic studies [l] . It was demonstrated that the rate dete rmining step in the formation of short double helices is a nucleation process of three base pairs. An appropriate kinetic description is given by an “all or none” process in which the only molecules in measurable concentration are fully base paired or single stranded. Related studies have been carried out with oligo d(A-T) [2]. The thermodynamics of the melting of a natural nucleic acid, tRNA

Destabilization of codon-anticodon interaction in the ribosomal exit site

& have been studied recently [3]. Evidence for double stranded regions in isolated tRNA fragments was reported in [4,5]. In the present paper the thermodynamic and kinetic behavior of half molecules from tRNAF& in the absence of magnesium is described. It was found that the double stranded structure of the halves is maintained and melts cooperatively. Similarly, the bimolecular recombination of the halves proceeds in a cooperative manner. From the negative apparent activation enthalpy of these reactions it is concluded that the formation of two or three base pairs is ratelimiting. The kinetic and thermodynamic data strongly support the clover-leaf model. 2. Materials and methods

A specific chemical chain scission of tRNA at 7-methylguanosine

The affinities of the exit (E) site of poly(U) or poly(A)-programmed Escherichia coli ribosomes for the respective cognate tRNA and a number of non-cognate tRNAs were determined by equilibrium titrations. Among the non-cognate tRNAs, the binding constants vary up to about tenfold (10(6) to 10(7) M-1 at 20 mM-Mg2+) or 50-fold (10 mM-Mg2+), indicating that codon-independent binding is modulated to a considerable extent by structural elements of the tRNA molecules other than the anticodon. Codon-anticodon interaction stabilizes tRNA binding in the E site approximately fourfold (20 mM-Mg2+) or 20-fold (10 mM-Mg2+), corresponding to delta G degree values of -3 and -7 kJ/mol (0.7 and 1.7 kcal/mol), respectively. Thus, the energetic contribution of codon-anticodon interaction to tRNA binding in the E site appears rather small, particularly in comparison to the large effects on the binding in A and P sites and to the binding of complementary oligonucleotides or of tRNAs with complementary anticodons. This result argues against a role of the E site-bound tRNA in the fixation of the mRNA on the ribosome. In contrast, we propose that the role of the E site is to facilitate the release of the discharged tRNA during translocation by providing an intermediate, labile binding site for the tRNA leaving the P site. The lowering of both affinity and stability of tRNA binding accompanying the transfer of the tRNA from the P site to the E site is predominantly due to the labilization of the codon-anticodon interaction.

Replacement of Y base, dihydrouracil, and 7-methylguanine in tRNA by artificial odd bases

The study of large fragments of tRNA has led to some insight into the mode of recognition of tRNA by its cognate aminoacyl tRNA synthetase [summaries 1, 2]. Interesting results were obtained also with respect to the physical properties of the fragments and their combinations [3 -5 ] . Most of the fragments had been obtained by partial digestion of tRNA with endonucleases, frequently at a modest yield [1, 2]. A completely specific and quantitative chemical method of chain scission had been described for preparing half molecules of tRNA Phe from yeast, wheat, and rat liver [6, 7]. We now describe a specific chemical method of chain scission at the m7G position of tRNA Phe (fig. 1), tRNA Phe (HC1), NaBH4 reduced tRNA Phe (HC1) from yeast, and the CCA halves of tRNA Phe from yeast and wheat. After excision of the alkali conversion product of m7G, the chain was split with aniline under mildly acidic conditions (fig. 2-4) . Pure fragments were isolated in 50 65% yield (fig. 5). The oligonucleotide analysis of a 30 nucleotide long fragment ranging from the lnTG position to the CCA end is reported (fig. 6). Combinations of this fragment with tire pG half of yeast tRNA Phe showed considerable acceptor activity (fig. 7) although 10 nucleotides are missing. The chain scission reaction should be applicable to the many tRNAs containing m 7 G.

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

Fluorescent dyes have been introduced into tRNA by reaction with the purine bases [ 1,2] , by coupling to the ribose moiety of the 3’-terminal adenosine [3,4] , by replacing this nucleoside by formycine [S] , and by non-covalent association [6-lo] . It was not possible by any of the four methods to attach a marker molecule with a high fluorescence quantum yield to a defined position without changes in the native structure of the tRNA and/or loss of biological activity. Such an attachment is highly desirable both for structural studies and for the investigation of tRNA-protein interactions [lo] . We therefore developed a method to replace the Y base of yeast tRNAPhe, which itself possesses a weak fluorescence, by the highly fluorescent compounds proflavine (PF) or ethidium bromide (EB). Some physical and biological properties of the modified tRNAs are described. The method is suitable also for the introduction of fluorescent bases into the positions of 7-methylguanine or dihydrouracil. The insertion of bases with other properties, e.g. metal binding, seems feasible.

Tertiary structure interactions of 7‐methylguanosine in yeast tRNAPhe as studied by borohydride reduction

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.

Mechanism of ribosomal translocation tRNA binds transiently to an exit site before leaving the ribosome during translocation

According to the recently published crystallographic analyses of yeast tRNAPhe the positively charged m7G in the extra loop contributes to the stabilization of the tertiary structure by hydrogen bonding to the Cl 3-G22 base pair of the dihydrouridine stem [l-4] . The possibility of an additional electrostatic interaction of m7G with the closely neighbouring phosphate of A9 has been noted [4]. We have been interested in m7G as a point of specific chemical modification and cleavage of tRNAPhe [5]. Previous [6,7] and new results of NaBH4 reduction experiments prompted us to investigate the accessibility of m7G in tRNAPhe under a number of conditions. In the present study aniline catalyzed chain scission [5,8] was used as a sensitive method for the detection of m7G modification by NaBHe. The fragments Phe l-45 and Phe 47-76 are formed in this reaction, which can also be used in a preparative scale. The NaB& modification of m7G does not affect the activity of tRNAPhe in a number of biochemical assay systems. At low ionic strength m7G in tRNAPhe is reduced rather slowly as compared to its reactivity in the fragment Phe 38-76. With increasing ionic strength, however, the rate of reduction of m7G in tRNAPhe is greatly accelerated and approaches the rate observed in the case of Phe 38-76. This result is direct