According to Wobble Rules, What Codons Should Be Recognized by the Anticodon 5ã¢â‚¬â²-icc-3ã¢â‚¬â²?

• Journal List
• Nucleic Acids Res
• v.31(22); 2003 Nov 15
• PMC275538

Nucleic Acids Res. 2003 November xv; 31(22): 6383–6391.

SURVEY AND SUMMARY: Roles of v-substituents of tRNA wobble uridines in the recognition of purine-ending codons

Kazuyuki Takai

^anePrison cell-Free Science and Technology Inquiry Center, ²Section of Applied Chemistry, Faculty of Engineering and ³Venture Business Laboratory, Ehime Academy, 3, Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan and ⁴Section of Biophysics and Biochemistry, School of Science, Academy of Tokyo, 3-7-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, ⁵Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Nihon and ^sixCellular Signaling Laboratory and Structurome Enquiry Group, RIKEN Harima Plant at Spring-8, 1-1-i Kohto, Mikazuki-cho, Sayo, Hyogo 679-5148, Nippon

Shigeyuki Yokoyama

^aneJail cell-Costless Science and Technology Research Heart, ²Department of Applied Chemistry, Faculty of Applied science and ⁱⁱⁱVenture Business concern Laboratory, Ehime Academy, 3, Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan and ^fourSection of Biophysics and Biochemistry, School of Science, University of Tokyo, 3-7-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Nippon, ⁵Protein Research Group, RIKEN Genomic Sciences Centre, ane-vii-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan and ⁶Cellular Signaling Laboratory and Structurome Research Group, RIKEN Harima Institute at Spring-eight, one-ane-i Kohto, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan

Received 2003 Jul 22; Revised 2003 Sep ten; Accepted 2003 Sep 20.

Abstract

Many tRNA molecules that recognize the purine-catastrophe codons but not the pyrimidine-ending codons have a modified uridine at the wobble position, in which a methylene carbon is attached directly to position five of the uracil ring. Although several models have been proposed concerning the mechanism by which the 5-substituents regulate codon-reading backdrop of the tRNAs, none could explicate recent results of the experiments utilizing well-characterized modification-deficient strains of Escherichia coli. Here, we first summarize previous studies on the codon-reading properties of tRNA molecules with a U derivative at the wobble position. So, we propose a hypothetical mechanism of the reading of the Thou-ending codons past such tRNA molecules that could explain the experimental results. The hypothesis supposes unconventional base pairs betwixt a protonated grade of the modified uridines and the G at the third position of the codon stabilized by 2 direct hydrogen bonds between the bases. The hypothesis as well addresses differences between the prokaryotic and eukaryotic decoding systems.

INTRODUCTION

During poly peptide biosynthesis, the ribosomes select right aminoacyl-tRNA molecules one-by-one by recognizing the anticodon triplet of the tRNA molecule that fits to the A-site codon triplet. Co-ordinate to the wobble hypothesis (one), when a tRNA molecule is recognized equally a correct one, the 3rd and second nucleosides of the anticodon (positions 36 and 35, respectively) form Watson–Crick base pairs with the first and second nucleosides of the codon, respectively, and the nucleoside at the starting time position of the anticodon (position 34) forms a Watson–Crick or a wobble base pair with the nucleoside at the tertiary position of the codon (position III) (i). The base of operations pairs allowed between position 34 and position 3 were assumed to be just those that could form two or more than than 2 direct hydrogen bonds between them with a pocket-sized displacement from the position for the Watson–Crick base pair. A U-G pair with the U at position 34 could exist formed with a small deportation of the uracil base of operations toward the major groove side, while it was known at that fourth dimension that uridines at position 34 are quite often post-transcriptionally modified.

Modified uridines constitute at position 34 of naturally occurring tRNA species are classified into 2 groups (Fig. ​ 1) (2). A modified uridine with a methylene carbon directly bonded to the C5 cantlet (xm⁵U) (Fig. ​ 1a) is often plant in tRNA species that recognize just the purine-ending codons. The xm⁵U nucleosides are frequently thiolated at position two (xm⁵sⁱⁱU) or methylated at the ribose 2′-hydroxyl group (xm^fiveUm). A modified uridine with an oxygen atom direct bonded to the C5 atom of the uracil ring (xo⁵U) (Fig. ​ oneb) is often institute in tRNA species that recognize the U-, A- and G-catastrophe codons.

Chemical structures of modified uridines found at position 34 of tRNA species. Symbols of the five-substituents are shown in parentheses.

In the nowadays paper, we use an asterisk to represent any substituent. For instance, U* represents any of the naturally occurring modified and unmodified uridines. In the same way, xm^vU* stands for any of the xm⁵U derivatives including xm⁵U, xm^vs²U and xm^vUm. Parts of the nucleoside symbols, such as xm⁵ and s^two, may also exist used to stand for the substituents, as in Effigy ​ ane. 5′-Nucleotides may exist symbolized such every bit pxo^vU. The position of a codon nucleoside may be shown in parentheses in Roman numerals, such as Thou(3), and the position of the anticodon nucleoside may exist shown in the same way in Arabic numerals, such every bit U(34).

The puckering equilibrium of the ribose band of the nucleosides in RNA molecules is generally biased to the C3′-endo form instead of the C2′-endo form, which is required for the germination of the typical A-course helices. This bias is besides observed in mononucleotides and nucleosides. In xm⁵U*, the ribose puckering is biased to the C3′-endo conformation to a greater extent than in unmodified U (iii–6). On the other hand, the puckering equilibrium of pxo⁵U is much shifted to the C2′-endo class (3). It was besides shown that a U in the C2′-endo conformation could basepair with some other U through two direct base–base hydrogen bonds by a model building report (three). Therefore, information technology was proposed that the modifications in xm⁵U* restricts, and the xo^v modification promotes, the formation of the U*(34)–U(Iii) pair (3). Information technology has been shown that the exchange of U(34) of the unmodified grade of Escherichia coli tRNA₁ ^Ser by mo^vU(34) enhances the in vitro reading of the UCU codon (7).

It is noteworthy that this theory of the regulation of codon recognition at the level of the dynamic conformation of the nucleotides is based on the wobble hypothesis proposed by Crick (1): two direct hydrogen bonds are required between the bases at positions 34 and 3. A mechanism that does not require the two direct base–base of operations hydrogen bonds has also been proposed to contribute to the codon reading and is named equally the 'ii out of 3' mechanism (8,ix) (meet below).

Recently, the physicochemical effects of the mnm⁵ and south² modifications were elucidated in detail by NMR structural analyses of anticodon stem–loop (ASL) oligonucleotides from Eastward.coli tRNA^Lys (10). The comparison of dissimilar ASLs with unlike modifications showed that the south² modification enhances the stacking of the bases in positions 35 and 36 onto the 3′ side of the anticodon to elevate the interaction of these bases with the get-go and 2d bases of the codon. The mnm^v modification also reduces the flexibility of the anticodon and contributes to 'preorganize' the anticodon into an A-course structure ready to interact with the codon in collaboration with the s^two modification. This clearly explained the effects of each modification on the misreading of the AAU/C codons past the tRNA observed in vivo under an Asn starvation condition (11), with the assumption that the misreading primarily depends on the 'two out of three' mechanism. Therefore, in this case, the 'two out of iii' mechanism may dominate over the wobble mechanism. It is reasonable that, in such cases, the decoding properties of the modification-scarce tRNAs could not be predicted from the conformational properties of the nucleotide at position 34.

On the other manus, the in vivo effects of the lack of each modification on the reading of the GAA/Grand codons by Eastward.coli tRNA^Glu with an mnm^vs²U at position 34 were also measured with the modification mutants (12). However, the data could not be explained completely even with the dynamic 3D structures of the ASLs (10), every bit described in detail beneath. This may mean that some unknown mechanism, different from the conformational regulation, has some contribution to the codon reading past the tRNAs with mnm^vsouthward²U(34).

In the present paper, we first summarize the known experimental results and theories on the effects of the xm⁵ modification and on other related subjects. So, we propose a physicochemical model of xm⁵U(34)–G(Three) pairing that could explain the in vivo effects of the mnm⁵ and s² modifications on the reading of the purine-ending codons.

EXPERIMENTAL FACTS AND THEORIES

Views from tRNA composition

Distribution and properties of xm ⁵ U*(34). In East.coli, mnm⁵s²U(34) is found in tRNA^Lys, tRNA^Glu and ane of the 2 tRNAs^Gln (13). tRNA₄ ^Leu and tRNA₄ ^Arg accept cmnm⁵Um(34) and mnm⁵U(34), respectively (6,14). Many eubacteria also accept the cmnm^five or mnm^five modification in Leu (UUA/G), Gln, Lys, Glu and Arg (AGA/G) tRNA species (13). At least Eastward.coli tRNA^Lys, tRNA^Glu and tRNA₄ ^Leu could read the G-ending codons: tRNA^Lys and tRNA^Glu are the single tRNA species for the amino acids (15), and a su6 strain in which only tRNA₄ ^Leu could read the UUG codon grows very well (half dozen,16). tRNA₄ ^Arg was suggested to read the AGG codon simply weakly (17), although the experimental results are not very conclusive: overproduction of tRNA_iv ^Arg might have caused undermodification, which might have led to overproduction of tRNA molecules that exercise not recognize the AGG codon; and the tRNA species that compete with tRNA_four ^Arg in their frameshifting assay were different between the assay of the AGA and AGG codons, which fabricated directly comparison of the activities on the dissimilar codons difficult. Instead, the higher up in vivo experiment clearly showed that the tRNA^Glu mutant with mnm⁵U(34) could read the GAG codon efficiently (12). Human being mitochondrial tRNA^Leu (UUA/G) and tRNA^Lys are also the single tRNAs for the codons and have τm⁵U(34) and τm⁵southward²U(34), respectively (eighteen).

Difference in prokaryotic and eukaryotic systems. The substituents in the xm⁵U derivatives in prokaryotes and eukaryotes are dissimilar. Prokaryotic tRNAs have derivatives of mnm⁵U, and eukaryotic tRNAs take those of mcm^fiveU or ncm^vU (13). Although many bacteria dispense with some tRNAs with C(34) that would read the CAG, AAG or GAG codon, all eukaryotes so far investigated take the C(34)-containing tRNAs for these codons (19). Therefore, it is possible that the eukaryotic xm^vU derivatives do not pair with G(Three). Information technology has likewise been suggested that eukaryotes cannot decode Grand-ending codons with tRNAs having a U derivative at position 34, based on the fact that they take at least a copy of an Ile tRNA factor with a T at the first position of the anticodon (20), although the postal service-transcriptional modifications of the U.s. are unknown (the tRNA would insert Ile for the Met codon if it could read the Thousand-ending codon). This deviation between prokaryotes and eukaryotes could exist ascribed to the difference in the ribosomes as well as the divergence in the tRNA modifications. As for prokaryotes, we focus on the modifications in eubacteria, as information on archaebacterial modifications is express.

Undiscriminating reading by mitochondria and mycoplasma tRNA species with U(34). In mitochondria and mycoplasmas, many family codon boxes (a codon box is a set of four unlike codons that have the get-go 2 bases in common, and if information technology specifies a single amino acid in the genetic lawmaking, it is a family box) are each translated past only 1 tRNA species with unmodified U(34) (21–24). Therefore, it was proposed that this kind of barnyard codon reading is based on the 'ii out of iii' mechanism. In some cases, this undiscriminating codon reading was shown to be less meaning in the separate codon boxes (a split codon box is a codon box that specifies more than one amino acrid) than in family boxes (eight,9,25).

Unmodified U

In vitro translation assay and properties of undiscriminating tRNA species with unmodified U(34). Information technology is important to understand the codon-reading properties of tRNA species with an unmodified U(34) before discussing the properties of the modified species. Near studies on such tRNA molecules utilise an in vitro translation system. It is well known that the discrimination of the third bases of the codons will exist ambiguous if simply ane aminoacyl-tRNA species is used in excess to introduce radiolabeled amino acid into proteins (26). This could be so even in a split codon box (27). Information technology is likewise known that the accuracy of in vitro translation is affected past the reaction conditions. For instance, pH and the concentrations of magnesium ions and polyamines could touch the fidelity of translation (28). Therefore, information technology is necessary to command the experimental weather condition advisedly. All the same, such assays have been used successfully to determine the relative efficiency of a tRNA species in reading a codon as compared with that of another competing tRNA species.

tRNA^Gly from Mycoplasma mycoides with U(34) is a unmarried tRNA species for the 4 Gly codons (29). This tRNA reads all the four Gly codons even in an in vitro translation system from E.coli. It has also been demonstrated that this undiscriminating codon reading requires a C at the commencement position of the anticodon loop (position 32) of the tRNA (thirty–33). Notwithstanding, C(32) is very often plant in tRNA molecules. Therefore, information technology likewise seems essential that the interaction of the 'two out of 3' is stiff enough to support such ambiguous reading (25). These results also suggested that tRNAs with U(34) behave differently in different situations concerning the discrimination of the third bases of the codons.

On the other hand, Mycoplasma capricolum has two Thr tRNAs with A(34) and U(34) (24). Although the tRNA with U(34) reads all the four Thr codons, the reading of the ACC codon is weak when the tRNA with A(34) is competing in an in vitro translation system from M.capricolum (34). The readings of the ACU and ACG codons by the tRNA with U(34) are as well weaker than those by the tRNA with A(34). Thus, codon preferences could be observed in this case. Therefore, some interaction betwixt U(34) and the third base of the codon contributes to the undiscriminating reading.

Every bit it seems that at that place is some confusion in some of the literature concerning the use of the terms 'wobble machinery' and 'ii out of 3' mechanism, we define the meanings within this paper as follows. The wobble machinery is a class of those mechanisms by which two or more direct hydrogen bonds are formed betwixt the bases at positions 34 and III while two Watson–Crick base pairs are formed between the last two positions of the anticodon and the first 2 positions of the codon. The 'two out of iii' mechanism is a class of those mechanisms by which less than two direct base–base hydrogen bonds are formed between positions 34 and III while 2 Watson–Crick base pairs are formed at the other ii positions. A 'two out of three' mechanism is one that satisfies the criteria for this item mechanism. Therefore, a '2 out of iii' machinery may involve some interaction without two direct base–base hydrogen bonds between positions 34 and Three. A 'base pair' in this paper ways a pair of bases with two or more than than two direct hydrogen bonds between them, unless mentioned otherwise. Some researchers use the term 'four-way wobble'. Even so, we do not use this term because it is for the mechanism of the ambiguous recognition of the four unlike codons but is not for the mechanism of recognition of individual codons.

Unexpected inefficiency in the reading of Thou-ending codons by unmodified tRNAs with U(34). Artificial unmodified tRNAs with U(34) have as well been investigated with an in vitro translation system. The in vitro transcript of E.coli tRNA₁ ^Ser reads the UCA codon one-half as efficiently as the fully modified molecules, but does not read the UCU and UCG codons (the UCU codon may exist recognized weakly) (35). A sample of the same tRNA prepared by chemical synthesis followed by enzymatic ligation had the same codon-reading properties, and the substitution of the U(34) by an mo^vU enhanced the reading of the UCU and UCG codons (7). The transcripts with base changes at the second and third positions of the anticodon into AA, UC and CU, respectively, also read the A-ending codons and did not read the Chiliad-ending codons (36). Thus, the U(34)–G(3) interaction is very weak at to the lowest degree with the structural context of the tRNA. As all of these unmodified tRNAs discriminate well the UCX codons, irrespective of C(32) that the tRNAs have, information technology is unlikely that any of the codon readings depends on a 'ii out of iii' mechanism. Therefore, U(34), which primarily assumes the C3′-endo form, cannot class a base of operations pair with G(III). Something other than the ribose puckering equilibrium should be different between U(34) and xm^fiveU*(34).

In vitro analyses of the effects of the xm^fiveU* modifications

A-site binding of ASL. Oligonucleotide-dependent ribosome-binding experiments accept been used about conveniently for the determination of codon-specificity of tRNAs. Withal, researchers should choose an experimental status that gives reasonable results, as some tRNAs bind strongly and the others demark weakly. Nevertheless, many researchers accept obtained reasonable results. Recently, 17mer ASL oligonucleotides were found to bind to the ribosomes. Although they mainly bind to the P site in the original techniques, the binding to the A site could also exist measured as the tetracycline-sensitive binding (37). With this technique, the furnishings of the mnm⁵sⁱⁱU modifications were investigated (37). The results clearly showed that the mnm⁵ and s² modifications enhance the A-site binding to the AAA and AAG codons, and that the efficiency of the A-site binding is always in parallel with that of the P-site bounden. The results from the P-site binding assay showed that the ASLs with U(34) bind only in the cases where the codon is from a family unit codon box. It is noteworthy that, although the GUG codon could bind the corresponding ASL with U(34) with a low affinity, the GCG, UCG and CCG codons did not bind the ASLs with a measurable affinity. This is consistent with the inefficiency observed for the reading of the Thousand-ending codons by the unmodified tRNAs with U(34) during the in vitro translation analysis mentioned above. Anyhow, the modifications are required for the U*(34)-containing ASLs to bind to the G-ending codons.

xm ⁵ U* from eukaryotes. An early piece of work has shown that a yeast tRNA^Glu specifically translates the GAA codon in an in vitro translation system from rabbit reticulocytes (38). This tRNA has mcm⁵s²U(34), and this may fit to the above idea that eukaryotes do not utilise the U*(34)–Chiliad(3) wobbling (20). This tRNA was also investigated by the conventional ribosome-bounden method (38). The results were consistent with those from the in vitro translation assay, while the source of the ribosomes was E.coli. Therefore, information technology may be the difference in tRNA but not that in the ribosomes that causes the difference in the codon specificity between eukaryotes and eubacteria at least in this case. It was also shown that yeast tRNA₃ ^Arg with mcm⁵U(34) does not recognize the K-catastrophe codon either (39). Therefore, the inability to recognize G(III) in tRNA^Glu may be independent of the sⁱⁱ modification.

In vivo experiments using modification mutant strains

Rates of translation of the GAA/Thou codons. The effects of the mnm⁵ and s² modifications on the rates for the translation of the GAA and GAG codons were measured with the use of well-characterized E.coli mutants of the mnm⁵due southⁱⁱU modifications (12). The rates at which the GAG codon was translated in the strains with mnm⁵due south²U(34), due south^twoU(34) and mnm⁵U(34) were vii.vii, ane.9 and 6.ii codons/south, respectively, and the rates of the reading of the GAA codon were 18, 47 and iv.5 codons/s, respectively. Therefore, the due south² modification of mnm⁵U elevates the reading of the GAA codon and has only a small issue on the reading of the GAG codon, and the mnm^five modification of s²U restricts the reading of the GAA codon and enhances the reading of the GAG codon. Although the level of the available undermodified aminoacyl-tRNA species in each mutant was not clear, the elongation rates during the translation of the whole lacZ coding sequence were most the same for these mutants. Therefore, we believe that the results are highly reliable. However, these results cannot be explained completely by the 3D structures of the ASL variants as described in detail below.

Results from frameshift assays. Brierley et al. (40) also utilized the modification mutants of East.coli to decide the in vivo correlation between the mnm⁵southⁱⁱU modification and frameshifting efficiency at a coronavirus frameshift site. In their assay, the frameshift efficiencies at the AAA/G codons in the modification mutant strains were measured. They tried to translate the results with the assumption that the frameshift efficiency should be negatively correlated with the stability of the codon binding by the tRNA^Lys species with different modifications. However, the frameshift efficiency may as well be affected by the efficiency of the tRNA molecules to shift to the AAA codon in the –1 frame, and, if this was the rate-limiting step of the whole process, the frameshift efficiency should take reflected the relative efficiency to bind to the –one frame codon as compared to the 0 frame codon. Therefore, interpretation of these results as related to the efficiency of the A-site codon bounden is quite difficult, as pointed out in other papers (x,12).

Misreading of pyrimidine-catastrophe codons. The same E.coli strains were used to analyze the efficiency of the misreading of the AAU/C Asn codons by the tRNA^Lys modification mutants under an Asn starvation condition (11). The results indicate that both mnm^five and sⁱⁱ modifications enhance the misreading. These results were unexpected, because the modifications were thought to restrict wobbling at that time (3), simply were rationalized when the NMR structures of the wild blazon and mutant ASLs were revealed in item (x) (see below).

Physicochemical aspects

Conformational preferences of uridine derivatives and the expected basepairing design. Equally described in a higher place, the conformation of xm^fivesⁱⁱU is biased to the C3′-endo course (3,4,41). This conformational preference is mainly due to the s² modification, which would heighten steric repulsion between the O2′ and the cantlet at position 2 in the C2′-endo grade. Therefore, 2′-O-methylation was also suggested to stabilize the C3′-endo form. The xm^v modification too contributes to the stabilization of the C3′-endo form (5,x). By contrast, pxo^fiveU is much more in its C2′-endo form than pU. Although the mechanism of the preference is not clear, it was suggested to be due to the interaction between the five′-phosphate and the oxygen cantlet of the xo⁵ substituent (3). With the C2′-endo form, U*(34) could basepair with U(Three) if the codon and the second and third positions of the anticodon are in the A form, every bit shown by a model edifice report. Therefore, it was proposed that the conformational brake into the C3′-endo form in xm⁵U*(34) should prevent mispairing with U(III) and stabilize the correct pair with A(Iii) (3).

As long as the A-type RNA is assumed in the other parts of the codon–anticodon duplex, U(34) could not form a base of operations pair with C(III) because of steric hindrance (41,42). Therefore, the C2′-endo form could non explain the reading of the C-ending codons by mitochondrial and mycoplasma tRNAs with U(34). Although the anticodon loop of Due east.coli tRNA^Lys was recently shown to have a remarkable flexibility (10), information technology is well known that the 2′-hydroxyl groups of the five nucleosides of the codon–anticodon duplex except the first one of the anticodon are hydrogen-bonded to the ribosome at the A site (43). Thus, information technology is reasonable to consider that these v nucleosides should exist in the C3′-endo form on the A site, no thing how flexible the conformation of the unbound anticodon loop is.

In the original hypothesis (3), the U*(34)-G(III) was idea to exist possible with both of the C2′-endo and C3′-endo forms. However, the above in vitro translation experiments (4,35,36) showed that the U(34)–1000(Three) pair with the C3′-endo grade of the U should be weak. Thus, the xm^vs²U(34)–G(III) pair should exist very weak, because the C2′-endo form of xm^vs^twoU should exist less stable than that of U and the S…H-Due north hydrogen bail required for the base pair should be weaker than the O…H-N bail required for the U-G pair. Therefore, the reading of the AAG and GAG codons by Eastward.coli tRNA^Lys and tRNA^Glu, respectively, could not be rationalized by this theory, as pointed out in an fantabulous review (44).

NMR structures of the modification variants of the tRNA ^Lys ASL. As described above, the physicochemical furnishings of the modifications in mnm⁵s²U in the tRNA^Lys ASL have been elucidated past NMR analyses (10). The southward² modification enhances the stacking of the '2 out of iii' onto the 3′-side of the anticodon, and the mnm^v modification decreases the flexibility of the loop.

As described higher up, the stabilization of the A-form construction of the anticodon resulted in the elevated misreading of the AAU/C Asn codons during the Asn starvation. This enhancement of the misreading could not be predicted from the conformational properties of the nucleotides at position 34 (11). The prediction implied that the misreading should depend primarily on the wobble machinery with the U*(34)–U(III) base pair, instead of the 'ii out of three' machinery. The fact that the AAC codons were also misread under the starvation status indicates that at least the misreading of the AAC codons depended on the '2 out of three' mechanism. It is likely that the conformational restriction into the C3′-endo class by the modifications reduced the efficiency of the misreading by the wobble mechanism to the extent that information technology was lower than that by the '2 out of three' mechanism.

The due south² modification could be predicted to enhance the reading of the GAA codon from its structural effects, and it did in the in vivo experiment. In the instance of the GAG codon, the substitution of O2 with a sulfur cantlet would destabilize the wobble base pair because the O2…H-North hydrogen bond would be substituted by a weak S2…H-Northward hydrogen bond, if any, while the stacking enhancement should more or less recoup for the destabilization (though this point is not described explicitly in the published textile). Therefore, the pocket-size outcome on the reading of the GAG codon observed in the in vivo experiment could be rationalized. On the other manus, the mnm⁵ modification should stabilize the interaction with A(III), every bit it should stabilize the 'preorganized' conformation. This contradicts to the in vivo data. Equally for G(III), if the mnm⁵due south²U(34)–G(III) base pair is formed with the C2′-endo grade of the mnm⁵s^twoU, so the mnm⁵ modification should reduce the reading of the G-ending codon because it should destabilize the C2′-endo class, which is again contradictory to the in vivo result. This may mean that the mnm⁵southward²U(34)–G(3) pair with the C3′-endo form of the mnm⁵due south^twoU is stabilized by the mnm⁵ modification through some unknown mechanism.

Thermodynamic analyses. Stabilities of RNA duplexes could be estimated by measuring the melting profiles of the duplexes. Many oligonucleotides containing modified nucleosides have been studied past this method. It should be noted that the contribution of a single base of operations pair to the stability of the duplex could not be defined considering a base pair should affect the neighboring base pair interactions. However, a sum of free energy parameters for all the pairs of neighboring two base pairs in the duplex, plus the parameters for the terminal base pairs and other constants, could be a practiced estimation of the stability of the duplex (45,46). Substitution of an A-U pair in the middle of an RNA duplex by a G-U pair would usually destabilize the duplex, and the gratuitous energy difference could exist estimated easily if the neighboring base pairs are known. In the same way, the effect of the substitution of a concluding A-U pair past a 1000-U pair could be estimated. However, this substitution turns out to be not-destabilizing or fifty-fifty stabilizing, in general, when the A and K are at the five′-ends of the duplexes and the Us are at the 3′-ends (46). Codon–anticodon duplexes with U(Three) are in a similar situation to the latter case, as position 34 is at the 5′-terminate of the codon–anticodon duplex. It has besides been observed that the substitution of A(34) by a Thou in a series of unmodified tRNAs raises, or does not change, the efficiencies to read the U-ending codons in an in vitro translation system (36). Therefore, the furnishings of a modification of U(34) could not be estimated from the stabilities of RNA duplexes with the modified uridines in the centre or at the 3′-stop of the duplexes. There have been no experiments in which the effects of the 5-substitution of uridines at the five′-end of RNA duplexes are estimated, although fifty-fifty this blazon of experiment would not necessarily be promising in terms of the estimation of the furnishings of the modification on tRNA codon recognition.

Model-building study (Lim's model). Lim and coworkers have proposed a model that explains the codon-reading patterns as related to the properties of the nucleosides at position 34 (42,47). In the model, U(34) could interact with U(III) and C(III) through h2o bridges, and a tRNA with U(34) reads all the 4 bases at position Iii. Therefore, the model may exist useful to predict codon preferences when the family unit codons are not fully discriminated. The mnm⁵ exchange would interruption some bonds needed for the water-bridged pairs, and this loss of the stabilizing interaction would non be compensated. Therefore, the modification should restrict the formation of the water-bridged pairs. However, it is obvious that this model does not take into account that the strength of the interaction between the get-go two codon positions and the last two anticodon positions could be changed by the due south² and xm⁵ modifications (10). Therefore, the model could not predict the in vivo furnishings of the modification (11,12).

A MODEL OF THE xm⁵U(34)–Grand(Three) BASEPAIRING

Here, we propose a model of the xm⁵U*(34)–G(III) pairing, by which the in vivo effects of each modification on the codon-reading rates and the prokaryote/eukaryote difference of the wobble dominion apropos the U*(34)–Yard(Three) pairing could be rationalized. The xm⁵U nucleosides from prokaryotes and mitochondria are derivatives of v-aminomethyluridine (xnm⁵U*), while those from eukaryotes are not (13). As for xnm⁵U*, the 5-substituent is likely to lower pK _a at the N3 position of the uracil band, because the positively charged nitrogen atom of the substituent should withdraw electrons from the uracil ring. Thus, xnm⁵U* may partially ionize under the physiological status. The ionized form of the nucleoside (xnm^fiveU*^-) could base pair with G(III) in two different configurations (Fig. ​ 2a and b). In the example of the 2-thiolated uridines, the ionization could confer a negative accuse on the sulfur atom and catechumen it to an efficient proton acceptor. The ionized form would be able to pair simply with G(III), and the neutral form, which may exist symbolized here as xnm^vU*⁰, could pair only with A(III). Both pairs could be formed with the C3′-endo conformation. Thus, the stabilization of the C3′-endo course by the modifications would stabilize both pairs. We suppose that the ionization should be partial. The relative efficiency in the reading of the G-catastrophe codon to that of the A-ending codon would non only depend on the degree of the ionization, merely would also depend on the difference in the intrinsic stabilities between the xnm^fiveU*^-(34)–G(Iii) and xnm⁵U*⁰(34)–A(3) pairs. Therefore, an xnm⁵U* could pair with G(III) more efficiently than with A(3) even when the neutral course is the major species. In the instance of mnn⁵s^twoU, the pairing in the neutral class with A(Three) may be all the same more than efficient, in total, than the pairing in the ionized form with G(III). The ionized modified uridine would not pair with U(III) or C(Iii). As the eukaryotic xm⁵ substituents practise not withdraw electrons as xnm⁵ may practise, the eukaryotic tRNAs practise not recognize the G-ending codons.

The proposed base pairs between a deprotonated modified U and a G proposed in the present study (a and b) and the conventional wobble U–G pair (c). (a) The proposed xnm⁵U*^- –G pair with the Watson–Crick configuration; (b) the alternative xnm^vU*^- –M pair with the xnm^vU*^- displaced toward the pocket-size groove side from the Watson–Crick configuration; (c) the conventional wobble U-G pair. The sulfur atom in (a) and (b) could exist substituted with an oxygen atom, and the negative accuse could exist delocalized within the π-electron system.

HOW THE MODEL FITS TO THE KNOWN FACTS AND THEORIES

Hammett equation and the known pK _a values for uridine and uracil derivatives. The p1000 _a values of a series of substituted compounds could be well predicted using the Hammett equation (48), as described in many organic chemistry textbooks. pK _a of a substituted molecule is predicted to be lower than the unsubstituted i by σ times ρ, where σ is a abiding specific to the substituent and its position, and ρ is a constant specific to the core acid. Equally judged from the known pK _a values for several uracil derivatives (U, 9.three; m⁵U, 9.7; and ane-methyl-5-bromouracil, 7.8) (49) and the σ values for the meta position (methyl, –0.07 and bromo, 0.39), ρ for uracil should exist positive (and ∼5).

The effects of substitutions at aromatic rings are mainly ascribed to 2 factors: inductive electron withdrawal and the resonance electron donation by the substituent. In the instance of the xnm⁵ substituent, the resonance effect may be small because of the methylene group directly attached to the uracil ring, and the inductive effect may be large because the nitrogen atom is protonated and is positively charged. Therefore, it is expected that the anterior result dominates over the resonance outcome, and the pM _a of mnm⁵U should be significantly lower than that of U. In fact, the pK _a values of cmnm⁵Um and cmnm⁵U have been measured to be viii.iii and viii.ii, respectively (half-dozen). As the negative accuse of the carboxyl group of the cmnm⁵ substituent may somewhat neutralize the electron withdrawing effect of the charged nitrogen, it is expected that the pM _a value for mnm⁵U is not ≤8.ii. Furthermore, as pK _a for s²U (viii.8) is lower than that for U by ∼0.five, it is expected that pThou _a for mnm⁵s^twoU should exist <8.

pK _a values for the uracil N3 position are lower in nucleosides than in the corresponding nucleotides, in general, because of the negative charge of the phosphate group. In fact, the measured values (pU, ix.7; pm^vU, 10.1; 5-bromouridine 5′-monophosphate, viii.i) (50) are higher than those of the corresponding nucleosides by ∼0.4. We have to be careful when comparing pK _a values measured under unlike conditions considering the issue of the charge of the phosphate may depend on the ion force and probably on the conformational preference of the nucleotide. However, it is still reasonable to assume that the pK _a value for pmnm⁵s²U may exist college than that of the nucleoside by ∼0.4.

The environment around the uracil ring on the decoding site of the ribosome may also affect the pYard _a values. However, from the recent results of the X-ray crystallographic analyses of the ribosome (51), nosotros could see that the charged group nearest to the showtime base of the anticodon of the A-site-bound tRNA is its 5′-phosphate, and that the altitude of the phosphate from the uracil ring may exist nearly the same as in the free nucleotide. Therefore, at that place is no reason at present to consider that the pG _a values on the ribosome are much higher than those in the corresponding nucleotides.

If the pK _a value of a molecule is eight.4, for instance, a 9% fraction of the molecule is ionized nether pH vii.4. Therefore, it is reasonable to assume that a considerable fraction of mnm⁵s²U in tRNA is ionized under the physiological status.

Two possible configurations. Two different configurations might be possible for the pair between xnm^fiveU*^-(34) and G(III) (Fig. ​ iia and b). Nosotros expect that the one with the Watson–Crick configuration (a) should be more stable than the ane with the wobble configuration in which the xnm^fiveU*^- is displaced toward the minor groove side (b). However, it is still possible that the latter configuration could contribute significantly. The pair may exist possible with the same ribose–phosphate conformation as in the Thousand(34)–U(3) wobble pair, which could exist stabilized by the stacking onto the neighboring base pair (two), and was shown to be no less stable than the A(34)–U(III) pair in a cell-gratis translation assay (36), as stated to a higher place. Thus, the wobble form of the xm⁵U*^-(34)–G(III) pair might be stabilized by the aforementioned machinery. In improver, every bit the sulfur atom of the wobble xnm⁵s²U*^-(34)–One thousand(3) pair should not participate in the hydrogen bonding interactions, the pair could be more stable than the pair with the Watson–Crick configuration. The possibility of the dual-way base pairing itself might also contribute to the enhancement of the reading of the G-ending codon.

Physicochemical and biological effects of the mnm ⁵ s ² U(34) modifications. As mentioned in a higher place, it is expected that the southward² modification of xnm^fiveU decreases the pK _a value. Therefore, the modification should promote the deprotonation. In addition, the s² modification should promote the stacking of the anticodon as in tRNA^Lys (ten) and stabilize the C3′-endo form of the nucleoside. If, for instance, the conformational effect stabilizes the pair by 5-fold and the fraction of the ionized form increased by the thiolation is 20%, then the full effect should be stabilization by iv-fold (though this may exist an oversimplification). Therefore, the xnm^fiveU-to-xnm^fives²U modification should enhance the reading of the A-catastrophe codon. This is consistent with the example of E.coli tRNA^Glu (12). Every bit for the One thousand-ending codon, the xnm^fives²U^-–Grand pair could be weaker in itself than the xmn^vU^-–Yard pair, considering at least i of the 2 possible base pair configurations in the quondam (Fig. ​ two) needs a hydrogen bond that involves the sulfur atom. Therefore, it is possible that this effect cancels the conformational furnishings. This is non contradictory to the fact that the s² modification of tRNA^Glu had only a small effect on the reading of the GAG codon in vivo (12). The experimental results likewise indicate that the reading of the GAG codon past the tRNA with mnm^vU(34) is more efficient than the reading of the GAA codon (12), and this could be reasoned if we assume that the mnm⁵U^-–1000 pair might exist intrinsically much more stable than the mnm⁵U⁰–A pair.

The mnm⁵ modification of southward^twoU would crusade the fractional ionization, which would destabilize the pair with A(III) and stabilize the pair with G(III), and the restriction of the flexibility of the anticodon, which would stabilize both pairs through stabilizing the C3′-endo form. Therefore, the pair with G(III) should be stabilized, while the prediction of the effects on the pair with A(III) is more difficult. The conformational event might exist smaller for the southward^twoU-to-mnm⁵s^twoU modification than for the U-to-mnm⁵U modification, as the southward²U-containing anticodon is already much biased to the preorganized conformation (10). On the other hand, the decrease in the fraction of the neutral form required for the pairing with A(Three) by the lowering of pG _a should exist larger in the presence of the s² modification, as s²U has a lower pThousand _a value than U. Thus, the mnm⁵ modification might exist more destabilizing as for the pair with A(III) in thiolated uridines than in non-thiolated uridines. In the case of tRNA^Glu (12), the effect through the deprotonation may have been larger than the conformational effect. As xnm^fiveU^-* could form no base pair with U(III) or C(III), the observed misreading of the AAU/C codons by tRNA^Lys during Asn starvation (11) should not have been due to the ionized class.

ii-Selenouridine derivatives. Leaner modifies mnm^fivesouth^twoU(34) into 5-methylaminomethyl-2-selenouridine (mnm^vSe^twoU), when selenium is bachelor (52). The pOne thousand _a value for this nucleoside is ∼seven.1, and a glutamate tRNA with mnm^vSe²U(34) binds efficiently to the GAG codon (53). Thus, it has been proposed that the ionized grade is responsible for the base pairing with Chiliad(Iii) (53). It seems that the depression pThou _a was considered to be mainly due to the two-seleno substitution, only the mnm^v substitution might also contribute.

xo ⁵ U. xo⁵U(34) has been proposed to recognize G(Iii) with the C2′-endo conformation (iii,7). If the pChiliad _a value for pxo⁵U is low enough, it would exist besides possible that xo⁵U(34) could also exist partially ionized. In fact, the pThou _a value for pmo⁵U has been measured to be viii.96 (fifty). The pThou _a values for the xo^vU nucleosides could also be predicted based on Hammett equation: pK _a for mo⁵U and ho⁵U could be predicted to be ∼8.7, using the σ values for the methoxy and hydroxyl substituents at the meta position (0.12) and the above-estimated ρ value for uracil. Therefore the predicted value is consequent considering the result of the 5′-phosphate. In cmo^fiveU, the pK _a could be higher than in mo^fiveU because of the negative charge in the carboxyl group. Thus, only a small fraction could be ionized in these nucleotides nether the physiological condition. In add-on, the C3′-endo conformation is destabilized in these nucleotides. Therefore, it may be reasonable to consider that the master mechanism for the G(III) recognition should be the formation of the neutral xo^vU(34)–G(3) pair with the C2′-endo form of xo⁵U.

Eukaryotic xm ⁵ U* nucleosides. In mcm^fiveU* and ncm⁵U* found in eukaryotic tRNAs (13,54), the substituents are unlikely to withdraw electrons also as the xnm⁵ group may practise. Therefore, it could be predicted that the tRNAs with the nucleoside could non pair efficiently with G(Iii). The experimental facts are as described in a higher place, and the G-ending codons in eukaryotes seem to exist recognized by tRNAs with C(34) in general (19,54). This could be an explanation for the prokaryote/eukaryote divergence of the wobble rule, if whatever, though it is still possible that the ribosomes are too dissimilar. It is possible that the tRNA modifications and ribosome functions have coevolved to optimize the translational function. If the present model is correct, it would mean that the eukaryotic ribosomes dispense with the C2′-endo conformation of U*(34) and a wobble pair with the anticodon base shifted toward the major groove, every bit eukaryotes do not have xo⁵U(34) (thirteen). Thus, it is possible that the eukaryotic ribosomes have lost the power to have eubacterial tRNAs with xo⁵U(34) for the reading of the U- and Thousand-ending codons.

TESTING THE MODEL

An obvious experimental test of the model is to measure the p1000 _a values of the modified uridines. This could disprove the model if the pK _a values for mnm⁵U* or τm⁵U* are non lower than ∼viii.v, which is unlikely considering the values for cmnm⁵U and cmnm⁵Um.

Another possible experiment may be to measure out the pH dependence of the relative efficiency of the U*(34)-containing tRNA in the reading of the Thou-catastrophe codon as compared to the efficiency of the tRNA with C(34) (35). However, the experiment could be difficult considering the overall fidelity in jail cell-free translation from E.coli depends on pH (28). Conventional ribosome-binding experiments and other advanced A-site bounden methods may also be possible (37,55).

Information technology is known that mnm⁵southward²U(34) in tRNA molecules could exist reversibly oxidized past iodine (56). Therefore, it may be possible to test the pH sensitivity of this reaction using prokaryotic and eukaryotic tRNA molecules, as the fraction of the ionized species should straight correlate with the reactivity. It may also be possible to gauge the pThousand _a values of xnm⁵U* in tRNA molecules by measuring the pH-dependence of the aminoacylation reactions catalyzed by the corresponding aminoacyl-tRNA synthetases. Fortunately, bacterial Lys-, Glu- and Gln-tRNA synthetases are in straight contact with position 34 when complexed with the cognate tRNA molecules (57–59).

It may besides be possible to measure out the efficiency of the reading of a codon with an inosine at the 3rd position. If the displacement of the uracil band of the modified uridine to the major groove side is possible, it is expected that the I(III)-containing codon also could exist recognized (come across Fig. ​ iic). On the other hand, if the ionization is responsible for the pairing with Chiliad(III), the codon with I(3) could not be recognized. Some other methods may also be possible that could determine whether the 2-amino group of G(III) participates in the base pair hydrogen bonding.

The 3D structure of the decoding site is emerging in particular in these years (43,51,lx,61). However, the position of the base of G(34) in the crystal was somewhat deviated from the normal wobble configuration. Therefore, it may still have some fourth dimension to localize the uracil ring of some of the modified uridines on the A site precisely plenty to tell the base pair configuration.

ACKNOWLEDGEMENTS

We thank Dr Mitsuo Sekine (Tokyo Institute of Technology) and Dr Kensaku Sakamoto (Tokyo Academy) for discussion. This work was supported in part by the Sumitomo Foundation, Tokyo, Nihon (no. 020764).

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