Conformational aspects and functions of trna

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1 Proc. Int. Symp. Biomol. Struct. Interactions, Suppl. J. Biosci., Vol. 8, Nos 3 & 4, August 1985, pp Printed in India. Conformational aspects and functions of trna T. MIYAZAWA* and S. YOKOYAMA Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan Abstract. In the process of protein biosynthesis, an aminoacyl-trna synthetase strictly recognizes the cognate amino acid and trna species. Spectroscopic and biochemical analyses have been made of a heterologous system of Thermus thermophilus glutamyl-trna synthetase and Escherichia coli trna Glu. The conformational difference between the initial complex and active complex has been observed, which is probably related with the strict recognition of aminoacyl-trna synthetase. trna species are post-transcriptionally modified at specific sites. Two types of modified uridine nucleosides have been found in the first position of anticodon, namely 5-hydroxyuridine derivatives (xo 5 U) and 5-methyl-2-thiouridine derivatives (xm 5 s 2 U). From the analyses of nuclear magnetic resonance spectra, the conformational characteristics of the two types of modified uridine nucleotides have been found to be remarkably different from each other. The conformational flexibility of xo 5 U nucleotides allow the multiple recognition of codons, whereas the conformational rigidity of xm 5 s 2 U nucleotides guarantees the recognition of correct codons. The modification of uridines in the first position of anticodon contributes to the correct and efficient translations of codons in protein biosynthesis. Keywords. Aminoacyl-tRNA synthetase; aminoacylation; codon-recognition; conformation of trna; modified uridine; nuclear magnetic resonance. Translation process in protein biosynthesis For any living organism, the protein biosynthesis is one of the most essential process for life. The genetic information, or the nucleotide sequence of messenger RNA is translated to the amino-acid sequence of protein. A codon of messenger RNA that specifies an amino acid, however, does not directly interact with the amino acid. There, a transfer RNA (trna) serves as an adaptor molecule. The X-ray crystal analyses have been made of yeast trna Phe and the molecular conformation of trna has been found to be an L-shaped structure (Kim et al., 1974; Robertus et al., 1974). In the translation process, each trna is covalently linked with the specific amino acid by the cognate enzyme, aminoacyl-trna synthetase. Subsequently on the ribosome, an aminoacyl-trna recognizes codons corresponding to the amino acid as linked to the 3'-terminal adenosine of trna. Therefore, the correct translation of the codon to the amino acid is due both to the strict recognition of cognate amino acid and trna species by the aminoacyl-trna synthetase and to the correct recognition of the codons by aminoacyl-trna species. * To whom correspondence should be addressed. Abbreviations used: NMR, Nuclear magnetic resonance; CD, circular dichroism. 731

2 732 Miyazawa and Yokoyama For the elucidation of the molecular mechanisms of such recognition in solution, a variety of spectroscopic methods may be applied, including analyses of nuclear magnetic resonance (NMR), together with fluorescence and circular dichroism (CD). In particular, for NMR analyses, however, thermostable samples of high purity are required. Accordingly, we have undertaken the purification of aminoacyl-trna synthetases from an extreme thermophile, Thermus thermophilus HB8. We have already succeeded in the purification, to homogeneity, of five enzymes specific to glutamate (Hara-Yokoyama et al., 1984), valine, isoleucine and methionine (Kohda et al., 1984), and phenylalanine. The subunit structures and molecular weights of these enzymes from this extreme thermophile are much the same as those of corresponding enzymes from Escherichia coli (Joachimiak and Barciszewski, 1980). Glutamyl-tRNA synthetase (GluRS) from T. thermophilus is a small monomer protein with a molecular weight of about 50,000. This thermophile enzyme efficiently aminoacylates E. coli trna species as well as T. thermophilus trna species. This GluRS from T. thermophilus is remarkably thermostable; in the presence of trna Glu and L- glutamate, 85 % of the aminoacylation activity is retained after incubation at 65 C for up to 9h (Hara-Yokoyama et al., 1984). Aminoacyl-tRNA synthetases from T. thermophilus are remarkably resistant even to protease digestion and accordingly are suitable for detailed conformation studies by NMR analyses (Kohda et al., 1984). Initial complex and active complex of GluRS trna Glu In the homologous system of T. thermophilus GluRS and trna Glu, the initial rate of aminoacylation is slightly enhanced as KCl is added up to 100 mm. By contrast, in the heterologous system of T. thermophilus GluRS and E. coli trna Glu, the initial rate of aminoacylation is reduced to about 10% as the KCl concentration is raised from mm. In order to examine the salt effect on the complex formation, the intensity change of tryptophan fluorescence of GluRS on addition of E. coli trna Glu has been observed. However, in the experimental condition of the aminoacylation reactions, the enzyme is largely bound to trna Glu and thus the inhibition of aminoacylation by KCl is primarily due to the salt effect on the conformation of GluRS trna Glu complex rather than to the dissociation of such a complex. Therefore, CD analyses have been made of the complex of T. thermophilus GluRS and E. coli trna Glu (Hara et al., 1983). E. coli trna Glu exhibits two CD bands in the region nm; the negative band at 335 nm is due to 5-methylaminomethyl-2-thiouridine residue in the first position of anticodon and the other negative band at 315 nm depends on the conformation of ribose-phosphate chain. These bands are both significantly affected by the complex formation with GluRS, indicating the conformational change of trna involving the anticodon moiety. However, on addition of KCl up to the concentration of 100 mm, the intensity change of the band at 335 nm is suppressed. Even in the presence of 100 mm KCl, nearly 95% of trna Glu is still bound to GluRS, as found from the fluorescence analysis. The CD spectrum of free trna Glu is not affected either by the addition of 100 mm KCl. Therefore, these CD data indicate that the conformation of the GluRS trna Glu complex changes upon addition of KCl. Similarly, for the aminoacylation of E. coli trna Glu by T. thermophilus GluRS, the Michaelis constants

3 Conformational aspects and functions of trna 733 for trna Glu and L-glutamate are both increased on addition of KCl (up to 100 mm). Thus, the spectroscopic data together with biochemical data suggest the two step process for the complex formation of GluRS with trna Glu. In the first step, GluRS and trna Glu form a weak inactive complex where relatively rigid parts of the enzyme and trna are in contact with each other, and the conformations of these two molecules are not significantly affected by such contact. The recognition of trna Glu by GluRS will not be very strict in such a complex. However, in the presence of KCl at low concentration, the initial complex of T. thermophilus GluRS and E. coli trna Glu undergoes a conformation change to an active complex, through some mutual adaptation of relatively flexible parts of the enzyme and trna. The conformation of the trna molecule, at least around the anticodon moiety is significantly affected. The strict recognition of trna Glu by GluRS is probably achieved by such complex formation in two steps (Hara et al., 1983). The formation of the complex of aminoacyl-trna synthetase and cognate trna in two steps is consistent with the temperature jump experiment for the system of yeast phenylalanyl-trna synthetase and trna Phe (Krauss et al., 1976). However, in the heterologous system of T.thermophilus GluRS and E. coli trna Glu, the formation of an initial (inactive) complex has been observed in addition to an active complex (Hara et al., 1983). Detailed NMR analyses of the conformations of these two complexes, in comparison, will be important for elucidating the molecular mechanisms as involved in the strict recognition of cognate trna species by aminoacyl-trna synthetase. Modification of uridine in the first position of anticodon In the translation process of protein biosynthesis, certain trna species recognize more than one codons and the number of trna species required for translating genetic codes on messenger RNA is appreciably smaller than 61, the number of amino acid codons. The third and second letters of anticodon of trna form Watson-Crick base pairs with the first and second letters, respectively, of codon. In the wobble hypothesis (Crick, 1966), uridine in the first position of anticodon is supposed to form base pair with adenosine or guanosine, but not with uridine or cytidine as the third letter of codon. Actually, however, uridine in the first position of anticodon is almost always modified after being transcribed from DNA (Nishimura, 1979).

4 734 Miyazawa and Yokoyama There are two types of modifications so far found in the first position of anticodon. One is 5-hydroxyuridine derivative (xo 5 U) and this group includes 5-methoxyuridine (mo 5 U, X = CH 3, from Bacillus subtilis) and 5-carboxymethoxyuridine (cmo 5 U, X = CH 2 COO, from E. coli). The other is 5-methyl-2-thiouridine derivative (xm 5 s 2 U) and this group includes 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 + U, X = NH CH 2 3, from E. coli) and 5-methoxycarbonylmethyl-2-thiouridine (X = COOCH 3, from yeast and mammal). Thus, a wide variety of 5-substituents have been found, which are different among organisms or specificities of amino acids (Nishimura, 1979). Modified uridines, xo 5 U, are found for trna species specific to valine, alanine, threonine and serine. Each of these amino acids has four degenerate codons terminating in uridine, cytidine, adenosine and guanosine. The trna species with xo 5 U recognize uridine in addition to adenosine and guanosine as the third letter of codon. By contrast, modified uridines, xm 5 s 2 U, are found in trna species specific to glutamine, lysine and glutamate. Each of these amino acids has two degenerate codons terminating in adenosine and guanosine. The trna species with xm 5 s 2 U primarily recognize adenosine, but not uridine or cytidine (Nishimura, 1979). The codon recognition patterns of trna species with xo 5 U are much the same, although there are a variety in size and in charge among the 5-substituents (O-X groups). The same is true for the codon recognition properties of trna species with xm 5 s 2 U, with a variety of 5-substituents (CH 2 X groups). Therefore, the direct interaction between the ribosome and 5-substituent of trna does not appear to be essential as far as these two types of modified uridines are concerned. Then, how can these two types of modifications affect the codon recognition in two distinct ways? In order to answer this question, a series of NMR analyses have been made of these modified uridines. Thus, the conformational characteristics of these two types of modified uridines have been found to be drastically different from each other, which are responsible for the two types of codon-recognition patterns (Yokoyama et al., 1983). Conformational characteristics of uridine nucleotides Uridine nucleotides in solution are flexible molecules, with the two puckering forms (C2 -endo and C3 -endo) of the ribose ring, the three rotamers (gg, gt and tg) about the exocyclic C4 -C5 bond and the two rotamers (G + and G ) about the C3 O3 bond (figure 1). For elucidating the conformations and conformer equilibria of such flexible molecules in solution, the lanthanide-probe NMR analysis is the most useful method (Inagaki and Miyazawa, 1981). The lanthanide-induced shift of resonance frequency of polar coordinates of the ith nucleus in the coordinate system of the principal magnetic axes with the lanthanide ion as the origin. On the other hand, Gd(III)-induced Relaxation rate of the ith nucleus is proportional to r i 6. For the combined analyses of such lanthanide-induced shifts and relaxation rates together with vicinal coupling constants, a new computer program (COFLEM) has been worked out. Thus, the structure parameters (conformations) and population parameters (conformer equilibria) together with Standard deviations may be obtained by the simulation of experimental data (Yokoyama et al., 1981).

5 Conformational aspects and functions of trna 735 Figure. 1. Local conformations of uridine 3 -phosphate. For uridine 3 -phosphate, the fractional populations of conformers have been determined. For the ribose-phosphate moiety, the C3 endo G form is the most abundant one (46 ± 6%) and the C2 endo G + form is the next abundant one (37 ± 14%). The population of the C2 endo G form is as low as 16 ± 13 %, and the population of the C3 endo G + form is negligible (3 ± 5 %), probably because of the steric repulsion between the phosphate group and the 5 CH 2 group. Thus, the interrelation between the ribose ring puckering equilibrium and the internal-rotation equilibria about the two exocyclic bonds has been elucidated by the lanthanide-probe method. The ribose ring in the C2 endo form preferentially takes the G + form about the C3 O3 bond, and the gg, gt and tg forms about the C4 C5 bond. By contrast, the ribose ring in the C3 endo form exclusively takes the gg form about the C4 C5 bond and the G form about the C3 O3 bond (Yokoyama et al., 1981). Such short-range interrelations affect the dynamic structural feature of the anticodon moiety of trna, as will be discussed later. The C2 endo form and C3 endo form are nearly equally abundant in unmodified uridine 5 -phosphate (pu). From the temperature dependence of the equilibrium constant, the enthalpy difference between the C2 endo form and C3 endo form has been found to be as small as 0 1 kcal/mol. However, in 5-methoxyuridine phosphate (pmo 5 U) and in 5-carboxymethoxyuridine phosphate (pcmo 5 U), the C2 endo form is more stable than the C3 endo form by as much as 0 7 kcal/mol. From the conformation

6 736 Miyazawa and Yokoyama analyses on related molecules, the significant stabilization of the C2 endo form in pxo 5 U is found to be due to the interaction of the 5 -phosphate group and the 5- substituent of the uracil base. By contrast, in 5-methylaminomethyl-2-thiouridine phosphate (pmnm 5 s 2 U), the C3 endo form is predominant and is remarkably more stable than the C2 endo form by as much as 1 1 kcal/mol. Such exclusive stabilization of the C3 endo form in pxm 5 s 2 U is primarily due to the interaction of the 2 OH group of the ribose ring and the bulky thiocarbonyl group (Yokoyama et al., 1979, 1983). Biological significance of post-transcriptional modification of uridine in the first position of anticodon of trna On the basis of the conformational characteristics of modified uridines, probable physical models have been found for the conformations of modified uridines as basepaired with the third letter of codons (Yokoyama et al., 1983). The base-pair with adenosine (figure 2a) is of the standard Watson-Crick type. The ribose phosphate moiety of the modified uridine in the first position of anticodon is set in the same conformation as the 2 -O-methylguanosine(34) residue of yeast trna Phe in crystal Figure 2. Base pairs of modified uridines with adenosine (a) and uridine (b) as the third letters of codons.

7 Conformational aspects and functions of trna 737 (Quigley et al., 1975). Thus, the conformation there is gg-c3 -endo-anti-g form, which has also been found to be stable by the lanthanide-probe NMR analysis in solution (Yokoyama et al., 1981). By contrast, for the formation of a short base-pair with uridine as the third letter of codon (figure 2b), the uracil base of the first letter of anticodon has to be displaced toward the codon. Surprisingly, such a displacement is in fact achieved by the conformational change from the gg C3 endo G form to the tg C2 endo G + form, without affecting the relative locations of the Ρ atom of the first letter and the C4 atom of the second letter of anticodon and the C1 atom of the third letter of codon. The combination of local conformations tg C2 endo G + is also found to be stable by the lanthanide-probe analysis. A probable physical model for the base pair with guanosine as the third letter of codon has also been found for the modified uridine in the C2 endo G+ form (Yokoyama, S. and Miyazawa, T., unpublished results). The post-transcriptional modification of uridine to 5-hydroxyuridine derivative (xo 5 U) stabilizes the C2 endo G + form as well as the C3 endo G form of the first letter of anticodon, because of the inherent stability of the C2 endo form in the xo 5 U unit itself. This modification makes the anticodon flexible and allows the recognition of uridine and guanosine in addition to adenosine as the third letter of codon. By contrast, the modification of uridine to 5-methyl-2-thiouridine derivatives (xm 5 s 2 U) further stabilizes the intrinsic conformation (C3 endo G form) of the first letter of anticodon, because of the inherent stability of the C3 endo form in the xm 5 s 2 U unit itself. This modification makes the anticodon even more rigid and thus allows the recognition of adenosine only, avoiding incorrect translation of codons of histidine, aspargine and aspartate in place of glutamine, lysine and glutamate. Thus, the biological significance of the two types of post-transcriptional modifications of uridine in the first position of anticodon is now found to guarantee the correct and efficient translations of codons, through the regulation of rigidity/flexibility of the anticodon moiety. References Crick, F. H. C. (1966) J. Mol. Biol., 19, 548. Hara, Μ., Yokoyama, S. and Miyazawa, T. (1983) International trna Workshop Abstract, Hakone, Japan. Hara-Yokoyama, Μ., Yokoyama, S. and Miyazawa, T. (1984) J. Biochem. (Tokyo), 96, Inagaki, F. and Miyazawa, Τ. (1981) Prog. Nucl. Magn. Reson. Spectrosc., 14, 67. Joachimiak, A. and Barciszewski, J. (1980) FEBS Lett., 119, 201. Kim, S. H., Suddath, F. L., Quigley, G. J., McPherson, Α., Sussman, J. L., Wang, A. H.-J., Seeman, N. C. and Rich, A. (1974) Science, 185, 435. Kohda, D., Yokoyama, S. and Miyazawa, Τ. (1984) FEBS Lett., 174, 20. Krauss, G., Riesner, D. and Maass, G. (1976) Eur. J. Biochem., 68, 81. Nishimura, S. (1979) in Transfer RNA: Structure, Properties, and Recognition (eds P. R. Schimmel, D. Soll and J. Ν. Abelson) (Cold Spring Harbor: Cold Spring Harbor Laboratory) p. 59. Quigley, G. J., Seeman, Ν. C., Wang, A. H.-J., Suddath, F. L. and Rich, A. (1975) Nucleic Acids Res., 2, Robertus, J. D., Ladner, J. Ε., Finch, J. Τ., Rhodes, D., Brown, R. S., Clark, B. F. C. and Klug, A. (1974) Nature (London), 250, 546. Yokoyama, S., Yamaizumi, Α., Nishimura, S. and Miyazawa, T. (1979) Nucleic Acids Res., 6, Yokoyama, S., Inagaki, F. and Miyazawa, Τ. (1981) Biochemistry, 20, Yokoyama, S., Watanabe, T., Murao, K., Ishikura, H., Yamaizumi, Ζ., Nishimura, S. and Miyazawa, T. (1983) International trna Workshop Abstract, Hakone, Japan.