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J. Biol. Chem., Vol. 277, Issue 19, 16391-16395, May 10, 2002

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Accurate Translation of the Genetic Code Depends on tRNA Modified Nucleosides^*

Connie Yarian^§, Hannah Townsend, Wojciech Czestkowski^¶, Elzbieta Sochacka^¶, Andrzej J. Malkiewicz^¶, Richard Guenther, Agnieszka Miskiewicz^¶, and Paul F. Agris

From the  Department of Biochemistry, North Carolina State University, Raleigh, North Carolina 27695-7622 and the ^¶ Institute of Organic Chemistry, Technical University, 90-924 Lodz, Poland

Received for publication, January 9, 2002, and in revised form, February 11, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Transfer RNA molecules translate the genetic code by recognizing cognate mRNA codons during protein synthesis. The anticodon wobble at position 34 and the nucleotide immediately 3' to the anticodon triplet at position 37 display a large diversity of modified nucleosides in the tRNAs of all organisms. We show that tRNA species translating 2-fold degenerate codons require a modified U₃₄ to enable recognition of their cognate codons ending in A or G but restrict reading of noncognate or near-cognate codons ending in U and C that specify a different amino acid. In particular, the nucleoside modifications 2-thiouridine at position 34 (s²U₃₄), 5-methylaminomethyluridine at position 34 (mnm⁵U₃₄), and 6-threonylcarbamoyladenosine at position 37 (t⁶A₃₇) were essential for Watson-Crick (AAA) and wobble (AAG) cognate codon recognition by tRNA at the ribosomal aminoacyl and peptidyl sites but did not enable the recognition of the asparagine codons (AAU and AAC). We conclude that modified nucleosides evolved to modulate an anticodon domain structure necessary for many tRNA species to accurately translate the genetic code.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

tRNA molecules play a significant role in the translation of the genetic code into protein sequences. The ribosome-mediated interaction of the mRNA codons with the anticodon of the tRNA results in the discrimination of cognate versus near-cognate and noncognate codons (1, 2). Cytoplasmic tRNA molecules contain ~80 posttranscriptional nucleoside modifications (3) with the largest diversity of these modifications located in the anticodon wobble at position 34 or immediately 3' adjacent to the anticodon triplet at position 37. Some of these nucleoside modifications appear to be necessary for efficient protein synthesis as determinants for aminoacylation (4, 5), which is important in maintaining reading frame (6) and necessary for codon recognition on the ribosome (7-9). We had previously reported that modified nucleosides in the anticodon loop of tRNA were essential for recognition of the lysine codons AAA and AAG. Specifically, 5-methylaminomethyluridine at position 34 (mnm⁵U₃₄),¹ 2-thiouridine at position 34 (s²U₃₄), and 6-threonylcarbamoyladenosine at position 37 (t⁶A₃₇) enabled codon recognition at the ribosomal peptidyl (P) site (7, 8). However, we had not previously compared the P site ribosomal contributions of these modified nucleosides in tRNA with contributions at the ribosomal A site, the ribosomal entry site for aminoacylated tRNAs during peptide elongation. In addition, it has not been shown whether the same tRNA modifications necessary for cognate codon recognition also restrict wobble recognition of near-cognate codons.

Crick's wobble hypothesis for codon recognition (10) has been revised (11-14) to include the influence of modified nucleosides. The wobble rules proposed by Crick are that U recognizes A and G, C recognizes G, A recognizes U, and G recognizes U and C. Lim and colleagues (11, 12) and Yokoyama and colleagues (13, 14) proposed that unmodified U₃₄ could recognize U and C in addition to A and G. The modifications of U₃₄ would restrict wobble recognition to A and G, but 5-oxyuridine modifications at position 34 such as 5-methoxyuridine (mo⁵U₃₄) and 5-carboxymethoxyuridine (cmo⁵U₃₄) would allow recognition of U as well as A and G (12). However, we had previously shown that the unmodified U₃₄ in completely unmodified tRNA, tRNA, and tRNA could not recognize A and G (7). Therefore, the need for a modified U₃₄ must be to restore and restrict wobble position recognition to A and G.

To identify tRNA species that require a modified U₃₄ for codon recognition and restrict wobble specificity of the first anticodon position, we assayed the ability of specific tRNAs to recognize cognate and wobble codons in the absence of their naturally occurring modifications at U₃₄. The 17 nucleotides of the anticodon stem and loop domain (ASL; Fig. 1) were used as a mimic of the entire tRNA molecule to assess codon recognition at the ribosomal A and P sites (7, 8, 15-17). The 30 S ribosomal P site has the highest affinity of all ribosomal sites for entire tRNA molecules and for ASLs. Thus, the P site is first occupied in vitro (18, 19), and binding to the A and P sites does not depend on the presence of initiation or elongation factors (16, 17). Here we provide experimental evidence that the need for a modified wobble position, U₃₄, directly correlates to the need for some tRNAs to discriminate at the third position of the codon. We also show that modified nucleosides in tRNA anticodon domain modulate codon recognition in the ribosomal A site similarly to the ribosomal P site.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

RNA Synthesis-- Unmodified and variously modified ASLs and 30-mer mRNAs were purchased from Dharmacon (Lafayette, CO) and Xeragon (Huntsville, AL) or synthesized using ribonucleoside phosphoramidite chemistries and methods as described previously (7, 8).

Ribosomal P Site Filter Binding Assay-- ASLs were 3'-³²P end-labeled as described previously (20). Small ribosomal subunits (30 S) were prepared from Escherichia coli MRE600 cells (21) and activated by incubating at 37 °C for 30 min in CMN buffer (80 mM potassium cacodylate acid, pH 7.5, 20 mM MgCl₂, 100 mM NH₄Cl). Ribosomal subunits were 70-95% active in all assays. ASLs were bound to the 30 S ribosomal P site as reported previously (7, 8, 15) with the exception that mixtures of ASL, 30 S subunit, and mRNA were incubated on ice for 1 h before being passed through nitrocellulose membranes. Binding curves were analyzed by one site nonlinear regression (GraphPad Prism) with yeast tRNA^Phe (Sigma) binding to poly(U) as the positive control.

Ribosomal A Site Filter Binding Assay-- ASLs were bound to 30 S ribosomal subunits with a procedure modified from that previously reported for determining only programmed P site binding (7, 8, 15). Small 30 S ribosomal subunits (10 pmol) and a 30-mer message consisting of 10 consecutive copies of the appropriate triplet codon either in the absence of tetracycline or in the presence of 250 µM tetracycline (Sigma) were incubated with 200 pmol 3'-³²P end-labeled ASL in 40 µl of CMN buffer plus 3 mM -mercaptoethanol. Reaction mixtures were incubated on ice for 1 h before being passed through nitrocellulose filters as described previously (7, 8). The total number of ASLs bound to 30 S ribosomal subunits was determined in the experiments without tetracycline. The number of ASLs sensitive to tetracycline was determined by subtracting the number of ASLs bound in the presence of tetracycline from the total number bound in the absence of tetracycline. The standard deviations for A site binding were determined by taking the square root of the sum of the squares of the standard deviations for the values in the absence of tetracycline and the presence of tetracycline. All values are averages and standard deviations of at least duplicate experiments.

Chemical Protection of 16 S rRNA Nucleotides-- E. coli 30 S ribosomal subunits (10 pmol) were programmed with an appropriate message in CMN buffer either in the absence of tetracycline or in the presence of 250 µM tetracycline in the absence of tRNA or in the presence of 200 pmol of ASL. The mixtures (40 µl) were incubated on ice for 1 h before chemical probing. Chemical modification of 16 S rRNA nucleosides was conducted by adding 2 µl of one-third dimethyl sulfate (Sigma) solution in 100% ethanol as described previously (7, 8). The dimethyl sulfate-modified 16 S rRNA was reverse-transcribed with a 5'-³²P end-labeled DNA primer complimentary to bases 1509-1530 to probe A1492 and A1493 and bases 824-845 to probe A794 and C795 in 16 S rRNA as described previously (7, 8). The reverse transcription products were electrophoresed by 6% PAGE/7 M urea.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Codon Recognition in the Ribosomal P Site-- Unmodified ASLs corresponding to the sequences of Lys_UUU, Glu_UUC, Gln_UUG, Arg_UCU, Ala_UGC, Ser_UGA, Val_UAC, and Pro_UGG tRNAs were chemically synthesized and assayed for their ability to bind to 30 S ribosomal subunits programmed with the respective cognate codons. The naturally occurring modifications in the anticodon domains of each of these tRNA species are listed in Table I. The unmodified Lys_UUU, Glu_UUC, Gln_UUG, Arg_UCU, and Ala_UGC ASLs did not recognize their cognate codons in the ribosomal P site (Table I). However, the unmodified Ser_UGA, Val_UAC, and Pro_UGG ASLs recognized their cognate codons (K_d = 600 ± 270 nM, 300 ± 100 nM, and 180 ± 60 nM, respectively) (Table I). The inability of an ASL to recognize its cognate codon on the ribosome was not a result of using only the isolated anticodon domain as a tRNA mimic. The completely unmodified transcripts of human tRNA, E. coli tRNA, and E. coli tRNA did not recognize their cognate codons on the ribosome (7), although completely modified native E. coli tRNA and tRNA recognized their respective codons (K_d = 70 ± 7 and 700 ± 300 nM, respectively) (7). With the incorporation of s²U₃₄ into the otherwise unmodified ASL, the ASL exhibited a relative binding constant of 105 ± 7 nM for the AAA codon and 200 ± 20 nM for the AAG codon and did not recognize the AAC or AAU codons (Table I).

Codon Recognition in the Ribosomal A Site-- To determine whether modified nucleosides have a contribution in the ribosomal A site, we assessed the ability of modifications previously shown to restore programmed ribosomal P site binding (7, 8) to restore programmed A site binding. The binding affinity of the ribosomal A site is ~100 times lower than that for the P site (18). Thus, to achieve A site binding to the 30 S subunit in vitro as described here, it is necessary to saturate the P site. An additional tRNA exit site on the ribosome, the E site, has also been identified for deacylated tRNAs (22), but the majority of the binding energy for this site appears to be in the 3'-terminal adenosine base of the entire tRNA molecule and the 50 S ribosomal subunit (23).

The binding of various modified ASL constructs to the programmed A site was inferred by determining the number of ASL constructs sensitive to the presence of tetracycline. In the presence of 250 µM tetracycline, the binding of tRNAs to the ribosomal A site is inhibited (24-27). In the presence of tetracycline, ASL constructs modified with s²U₃₄, mnm⁵U₃₄, or t⁶A₃₇ individually and with mnm⁵U₃₄ and t⁶A₃₇ combined (Table II) bound AAA-programmed 30 S ribosomal subunits effectively (Table II) but measurably lower than the binding observed in the absence of tetracycline. The difference in binding was attributed to binding at the tetracycline-sensitive ribosomal A site (Table II). In addition, ASL-s²U₃₄ and ASL-mnm⁵U₃₄ t⁶A₃₇ recognized the AAG codons in the ribosomal A site (Fig. 1). Therefore, modifications that restored P site ribosomal codon recognition also restore codon recognition to the ribosomal A site.

View this table: [in this window] [in a new window]   Table II Ribosomal A and P-site relative binding efficiencies of unmodified and variously modified ASL constructs

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Secondary structure of unmodified and variously modified ASL. The chemical structures of the modified nucleosides site-specifically incorporated into the unmodified ASL are displayed. The terminal base pair was changed from the naturally occurring A27-U43 to G27-C43 for increased yield in chemical synthesis (7, 8). Although the naturally occurring modifications forASL are mcm5 s2U34 and ms2t6A37, the combinations of these modifications cannot withstand the chemical synthesis procedure. Therefore, mnm5U34 and t6A37 naturally occurring modifications of tRNA species from different organisms and s2U34 were incorporated.

Chemical Protection of 16 S rRNA Nucleotides-- To confirm that we were observing A site binding in the absence of tetracycline but not in the presence of tetracycline, we monitored the altered chemical reactivity of 16 S rRNA bases A1492, A1493, A794, and C795 in the presence of the variously modified ASL. When tRNA is bound in the ribosomal A site, A1492 and A1493 are protected from chemical modification (28). Conversely, A794 and C795 are protected when tRNA is bound in the ribosomal P site (28). ASL-mnm⁵U₃₄t⁶A₃₇ and ASL-mnm⁵U₃₄ protected 16 S rRNA bases A1492 and A1493 from chemical modification in the absence of tetracycline but not in the presence of tetracycline (Fig. 2, lanes 3-6). The protection of A site bases was not evident when unmodified ASL was present (Fig. 2, lanes 7 and 8). The codon binding of the completely unmodified ASL both in the presence and absence of tetracycline was almost below detection, confirming only minimal codon recognition at either ribosomal binding site (Table II and Fig. 2). To ensure that the presence of tetracycline did not affect P site binding, we monitored the protection of A794 and C795. In the absence of tRNA or in the presence of the unmodified ASL, the ribosomal bases A794 and C795 were not protected. In the presence of ASL-mnm⁵U₃₄t⁶A₃₇ and ASL-mnm⁵U₃₄, the P site rRNA bases were protected both in the absence and in the presence of tetracycline (Fig. 2). These results confirmed that tetracycline was inhibiting A site binding and not influencing P site binding. Therefore, the ability of modified nucleosides to restore codon recognition at the ribosomal P site is similar to the ability at the ribosomal A site.

View larger version (79K): [in this window] [in a new window]   Fig. 2.   Protection of 16 S rRNA bases from chemical modification. The 30 S ribosomal subunits programmed with poly(A) were incubated with no tRNA (lanes 1 and 2), ASL-mnm5U34 t6A37 (lanes 3 and 4), ASL-mnm5U34 (lanes 5 and 6), or the unmodified ASL (lanes 7 and 8) either in the absence (lanes 1, 3, 5, and 7) or presence (lanes 2, 4, 6, and 8) of 250 µM tetracycline. The reactions were chemically treated with dimethyl sulfate followed by reverse transcription of the isolated 16 S rRNA. The 16 S rRNA bases A1492 and A1493 are protected from chemical modification when tRNA is bound in the ribosomal A site (top); A794 and C795 are protected when tRNA is bound in the ribosomal P site (bottom).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The work reported here provides experimental evidence for the role of tRNA modified nucleosides in maintaining accurate recognition of the genetic code. Many tRNAs can recognize more than one codon because of the ability of the anticodon wobble at position 34 to wobble to recognize the third position of the codon. These data indicate that modification of U₃₄ is the biochemical mechanism by which tRNA molecules accurately differentiate codons from multiple amino acid codon boxes. Multiple amino acid or mixed codon boxes refer to 2- and 3-fold degenerate codons that specify more than one amino acid by only a difference in the third base (Table III). We have found that the tRNAs that have a U₃₄ and translate codons from mixed codon boxes rely on the U₃₄ modification to enable recognition of A or G in the third position of the codon. tRNA, tRNA, tRNA, and tRNA translate codons from multiple amino acid codon boxes, and the corresponding unmodified ASLs did not recognize their cognate codons (Table I). tRNA, tRNA, tRNA, and tRNA all translate codons from single amino acid codon boxes, and all of the corresponding unmodified ASLs with the exception of ASL recognized their cognate codons, although with different affinities (Table I).

If a modified U₃₄ is necessary for tRNAs that translate codons from mixed codon boxes to recognize A and G, the modification may also restrict the recognition to A and G. Previously, we reported that the incorporation of the s²U₃₄ modification into the otherwise completely unmodified ASL restored recognition of the lysine codons AAA and AAG (7). Whereas this modification restored recognition of the two lysine codons AAA and AAG, it is necessary that the s²U₃₄ modification does not wobble to read AAU and AAC asparagine codons. We assayed the ability of the U₃₄ modification, s²U₃₄, in tRNA to restrict wobble recognition to A and G. With the incorporation of s²U₃₄ into the otherwise unmodified ASL, the ASL exhibited a relative binding constant of 105 ± 7 nM for the AAA codon and 200 ± 20 nM for the AAG codon and did not recognize the AAC or AAU codons (Table I). These experimental data support the work of Yokoyama et al. (14) and the model proposed by Lim and Curran (12) that modifications of U₃₄ are used in tRNAs that translate codons from multiple amino acid codon boxes to not only recognize A and G in the third position of the codon but also to negate the recognition of U and C. The 2-thiopyrimidine nucleosides and nucleotides are predominantly found in the C3'-endo conformation to recognize adenosine and to a lesser extent guanosine as the third nucleotide of the codon (14). It is absolutely essential that tRNAs reading from multiple amino acid codon boxes be able to discriminate the third position of the codon to ensure translational fidelity.

The cmo⁵U₃₄ modification is present only in tRNAs that read 4-fold degenerate codons. There is no requirement for these tRNAs to discriminate at the third position of the codon, and we have found that these tRNAs do not require a modified U₃₄ for cognate codon recognition. For example, unmodified ASL recognized the valine codons GUA and GUU with high affinity (K_d = 300 ± 100 and 260 ± 20 nM, respectively) and displayed low but measurable recognition of the valine codons GUC and GUG (Table I). Therefore, the unmodified U₃₄ in ASL efficiently recognized an A as the third nucleotide in the codon and surprisingly displayed high affinity to the codon with a U at the third position, a wobble recognition not originally proposed by Crick. It is reasonable that the incorporation of the naturally occurring position 37 modification, m⁶A₃₇, into ASL would enhance the recognition of the GUG codon. We had previously reported that the incorporation of both the position 34 and 37 modifications in tRNA enhanced the recognition of the AAG codon, whereas the singly modified ASL either at positions 34 or 37 did not recognize the AAG codon (8). Although the unmodified U₃₄ in some tRNAs with a naturally occurring cmo⁵U₃₄ modification may be able to recognize A, G, U, and C, the modification of cmo⁵U₃₄ may restrict the reading of C. Proton NMR analyses indicate that the cmo⁵U modification takes the C2'-endo form as well as the C3'-endo form to recognize uridine in addition to adenosine and guanosine as the third base of the codon (14).

tRNA was the only unmodified ASL, which we assayed, that has a naturally occurring cmo⁵U₃₄ modification and did not recognize its codon (Table I). Interestingly, it is also the only anticodon sequence we have assayed that has the cmo⁵U₃₄ modification without a naturally occurring position 37 modification (Table I). Therefore, the cmo⁵U₃₄ modification may be necessary to restore codon recognition to ASL, whereas the other ASLs with cmo⁵U₃₄ (ASL, ASL, and ASL) recognized their Watson-Crick base pairing codons but probably require modifications of either position 34 or both positions 34 and 37 to wobble.

Although contributions of anticodon tRNA-modified nucleosides have been established (6-8, 13), we had not previously compared the ability of the anticodon stem and loop constructs to recognize their cognate codons at the ribosomal P and A sites. The ribosomal position that first discriminates cognate from near-cognate and noncognate tRNAs during protein elongation is the ribosomal A site. Previous work has reported contributions of modified nucleosides in the ribosomal A site but not with the site-specific modified ASL constructs. It has been proposed that a hypomodified tRNA may decrease the rate by which the tRNA is recruited to the A site (6), that a position 37 modification enhances proofreading in the ribosomal A site (29), and that position 37 modifications influence the in vivo aminacylated tRNA selection in a tRNA-dependent manner (9). We determined that modified nucleosides have a contribution in the ribosomal A site similar to our previous observations of codon binding in the ribosomal P site. The modifications previously shown to restore programmed ribosomal P site binding (7, 8) restored the programmed A site binding.

The x-ray crystallographic structures of the 30 S ribosomal subunit (1) demonstrate that in forming the codon/anticodon duplex in the ribosomal A site, ribosomal nucleotides A1492 and A1493 flip out of the internal loop of ribosomal helix 44. In addition, the ribosomal nucleotide G530 switches from the syn-conformation to anti-conformation. In these new conformations, A1492 and A1493 interact with the first and second base pair in the minor groove of the codon/anticodon helix, and G530 interacts with the second position of the anticodon and the third position of the codon (1). Thus, the ribosome discriminates cognate from near-cognate and noncognate tRNA at two positions of the anticodon/codon duplex corresponding to the first and second positions of the codon base pairing with the third and second positions of the anticodon. The wobble position of tRNA (position 34) appears to be more suited to accommodate other geometries (1). Because the unmodified base sequences of some tRNA molecules do not bind their cognate codons in the ribosomal A site, we postulate that A1492 and A1493 are unable to interact with the minor groove of the codon/anticodon helix, and therefore, these tRNAs do not remain on the ribosome.

Studies on the human tRNA (30), human ASL (31), and E. coli ASL (32) have shown that the modified nucleosides in the anticodon domain of tRNA^Lys species with the UUU anticodon are necessary for the biochemical and structural characteristics of this molecule. The single incorporation of t⁶A₃₇, 3' adjacent to the anticodon triplet, inhibited base pairing across the anticodon loop (31), and the combination of t⁶A₃₇ with mnm⁵s²U₃₄ restored the characteristic U-turn in the anticodon, resulting in the canonical stacked anticodon triplet (30, 32). Lim and Curran (12) have proposed that the correct codon/anticodon duplexes are those whose formation and interaction with the ribosomal decoding center are not accompanied by uncompensated losses of hydrogen and ionic bonds. Therefore, to distinguish cognate from errant anticodon/codon duplexes, the ribosome must sterically restrict duplexes such that only cognate complexes form fully compensating bonds in the decoding center (12). Modified nucleosides appear to be necessary for the sequences of some tRNAs to create an anticodon loop that enables A1492 and A1493 to interact with the minor groove of the codon/anticodon helix without uncompensated losses of hydrogen and ionic bonds.

The modified nucleosides in the anticodon loop of some tRNAs modulate codon recognition in the ribosomal A and P sites. We have shown that the need for a modified U₃₄ directly correlates to the need for the tRNA to discriminate at the third position of the codon. Lim and Curran (12) have suggested that the requirement for modifications at position 34 may have arisen from evolutionary pressure to prevent errors in codon recognition (12). Primitive codes may have specified fewer amino acids, and the mixed codon boxes may have originally specified single amino acids. Restricting wobble at tRNA position 34 would have functionally split some of the codon boxes, allowing the incorporation of more amino acids into protein sequences (12). We have also found some ASL sequences that have an unmodified G or C at position 34, and a modification at position 37 also appear to require modified nucleosides (data not shown). Position 37 modifications may be involved in preventing frame-shifting (13) and probably have arisen from an evolutionary pressure to maintain correct translation of the genetic code. Modified nucleosides in the anticodon domain of tRNAs appear to be a means of expanding the complexity of mRNA molecules that interact with this region of the tRNA. The diversity of modified nucleosides at positions 34 and 37 may create an anticodon triplet unique enough to be specifically identified by the cognate aminoacyl-tRNA synthetase while maintaining an anticodon loop structure that allows a ribosome-mediated anticodon/codon discrimination during protein synthesis.

	ACKNOWLEDGEMENTS

We acknowledge Winnell Newman (North Carolina State University Nucleic Acids Facility) for expertise in RNA synthesis and Dr. Paul Wollenzien, his laboratory, and Dr. Stanislov Kirillov for assistance in ribosomal binding assays. We thank Drs. James Curran, James Brown, and Venki Ramakrishnan for editorial comments.

	FOOTNOTES

^* This research was supported by National Science Foundation Grant MCB9986011 and National Institutes of Health Grant GM23037 (to P. F. A.) and the Polish Committee for Scientific Research Grant 7T09A01721 (to A. J. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Present address: Dept. of Biochemistry and Molecular Biophysics, Washington University St. Louis, Box 8231, 660 S. Euclid Ave., St. Louis, MO 63110.

To whom correspondence should be addressed: Dept. of Molecular and Structural Biochemistry, North Carolina State University, 128 Polk Hall, Box 7622, Raleigh, NC 27695-7622. Tel.: 919-515-6188; Fax: 919-515-2047; E-mail: paul_agris@ncsu.edu.

Published, JBC Papers in Press, February 22, 2002, DOI 10.1074/jbc.M200253200

	ABBREVIATIONS

The abbreviations used are: mnm⁵U₃₄, 5-methylaminomethyluridine at position 34; s²U₃₄, 2-thiouridine at position 34; t⁶A₃₇, 6-threonylcarbamoyladenosine at position 37; mo⁵U₃₄, 5-methoxyuridine; cmo⁵U₃₄, 5-carboxymethoxyuridine; P, peptidyl; ASL, anticodon stem and loop.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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