Anticodon

 The anticodon of transfer RNA (tRNA) is in the central part of the linear RNA sequence, at positions 34, 35, and 36. This triplet of nucleotides forms transitory base pairs with three nucleotide codons of the messenger RNA (mRNA) during protein biosynthesis. These codon/anticodon interactions enable the mRNA to direct the order of incorporation of amino acids into the polypeptide chain. These interactions occur on the small subunit of the ribosome. The presence of the anticodon in the seven-membered central loop of tRNA led to the designation of this subdomain as the anticodon loop. The anticodon stem plus the loop constitute the anticodon arm (stem and loop), which contributes to one branch of the L-shaped 3D structure of tRNA. In this structure, the anticodon is about 80 Å from the amino acid acceptor end of the tRNA. Besides its fundamental contribution to translation of the genetic information through complementary pairing to the codon, the anticodon of tRNA may contain important identity elements, recognized by cognate aminoacyl-tRNA synthetases, which lead to specific aminoacylation of the tRNA. Besides the anticodon, other members of the loop and of the nearby stem, including modified nucleotides, also contribute both to the efficiency and specificity of aminoacylation.

Codon–anticodon interaction occurs basically through classic Watson–Crick base pairs. However, additional types of interactions allow wobble to occur between the third base of the codon and the first base of the anticodon (1). The original wobble rules suggested that the first nucleoside of the anticodon can pair with more than one nucleoside at the third position of the codon. Thus, anticodons with a U at the first position could interact with codons having either A or G at the third position. Those presenting a G at position 34 could interact with codons terminating with U or C. More interestingly, tRNA presenting an inosine (deaminated adenosine) at position 34 could recognize codons terminating with either C, U, or A. For example, yeast tRNAAla, anticodon 5′-IGC-3′, interacts with three codons: 5′-GCC-3′, 5′-GCU-3′, and 5′-GCA-3′.

Numerous data accumulated over the years led to revised wobble rules (2, 3), which reflect new possible interactions between classic bases and take into account the vast occurrence of modified nucleosides in the anticodons of tRNA, especially those at the wobble position (residue 34, first position of the anticodon). One minor tRNA, Escherichia coli, tRNA^Ile2, has a methionine anticodon CAU. However, in the mature tRNA, C34 is modified by covalent attachment of lysine on position 2 of the pyrimidine ring, converting it to lysidine or a “k2C” base. Lysidine at the wobble position leads to recognition of the isoleucine-specific codon 5′-AUA-3′ instead of the methionine codon AUG. Interestingly, this modification is also required for specific recognition of the tRNA by isoleucyl-tRNA synthetase (see discussion below). Other examples of modification at position 34 that contribute to the specificity of codon–anticodon recognition have been reported. This is also true for nucleotide 37, at the 3′ side of the anticodon triplet. Many different modified purine nucleosides have been identified at this position, and these modifications contribute to the fidelity of protein synthesis. For example, hypermodification of A to i6A (N-6-isopentenyladenosine), t6A (N-6-threonylcarbamoyladenosine), and their derivatives, stabilizes the relatively weak A-U and U-A base pairing between the third position of the anticodon and the first position of the codon. The presence of the hypermodified nucleoside ms2i6A (N-6-(D-2-isopentenyl)-2-methylthioadenosine), in contrast, prevents codon misreading by E. coli tRNA^Phe. Modifications introduce conformational flexibility or rigidity that restrict or enlarge the number of potential base pairs. Thus the molecular mechanism by which modified bases alter codon recognition are largely structural in nature.

UAG (amber), UAA (ocher), and UGA (opal) codons do not code for an amino acid, because there are no tRNA with corresponding anticodons. Known as nonsense codons, they normally signal termination of translation, but such codons can be created by mutation, when they cause premature  termination of protein synthesis. Interestingly, nonsense suppressor tRNA, with mutations in the anticodon, can recognize and “suppress” nonsense mutations by inserting a specific amino acid during translation (4). The first suppressor tRNA identified was a tyrosine-specific tRNA in which a single base substitution converted the anticodon from GUA (Tyr) to CUA (amber). Conversion of a classic tRNA to an efficient suppressor RNA may also involve mutations outside the anticodon. Suppressor tRNA have been used as a tool in the search of tRNA identity elements in vivo and as a way of inserting desired amino acids at specific sites in proteins.

 The anticodon triplet specifies which amino acid the tRNA will insert in response to a codon and is, of course, directly correlated with the amino acid bound to the -CCA end. A simple analysis of the genetic code, however, reveals that the anticodon is not a common signal for synthetase recognition. For example, there are six different serine codons and, consequently, the potential for six different anticodon sequences in serine-specific tRNA. This great variability in isoacceptor tRNA precludes a common signal in the anticodon for recognition by seryl-tRNA synthetase, but it is clear that anticodon nucleotides, as well as other nucleotides within the anticodon loop, are critical for which amino acids are charged by aminoacylation (5-10).

Single-point mutations at any anticodon nucleotide of yeast tRNA^Phe or yeast tRNA^Asp lead to severe losses in aminoacylation efficiency (11, 12). A simple anticodon switch changes the aminoacylation specificity of methionine and valine tRNA (13). Depending on the tRNA, one, two, or three anticodon nucleotides may be involved in tRNA specificity. Position 35 is required for charging of arginine, both 35 and 36 are important for valine or threonine, and 34, 35, and 36 are involved in specificity for Asn, Asp, Cys, Gln, Ile, Lys, Met, Phe, Trp, and Tyr. In E. coli tRNA^Gln, residue 35 is of greater importance than residues 34 and 36. In yeast tRNA^Asp, nucleotides 34 and 35 contribute more than do nucleotide 36. Transplantation of anticodons into noncognate tRNA results in increases of four to six orders of magnitude in aminoacylation of the chimeric tRNA with the amino acid corresponding to the transplanted anticodon. Since it does not preclude aminoacylation of the chimeric tRNA with the original amino acid, however, it is clear that additional identity elements elsewhere in the tRNA structure dictate amino acid specificity. The extent and the relative contribution of the different nucleotides of the anticodon to this specificity varies. The losses in specificity for yeast tRNA^Asp are about 500-fold, while those for E. coli tRNA^Val   are 100,000-fold.

Other nucleotides within the anticodon domain often contribute to amino acid specificity. Residue 38 is involved in yeast tRNA^Asp, and both residues A37 and U38 are involved in E. coli tRNA^Gln. An original approach to anticodon domain function uses the in vitro synthesis of minihelices (shortened forms of tRNA) mimicking isolated anticodon stem and loop domains. These minihelices possess identity elements and were tested for stimulation of aminoacylation rates of the acceptor stem as substrate and/or for inhibition of full-length tRNA charging (14). Yeast valyl-tRNA (15) and E. coli isoleucine-tRNA (16) anticodon minihelices stimulated, up to threefold, the aminoacylation of an acceptor arm minihelix by cognate aminoacyl-tRNA synthetase. The isolated anticodon domain of tRNAf^Met binds to E. coli methionyl-tRNA synthetase (17).

 The anticodon domain is further implicated in discrimination between initiator and elongator forms of tRNA (18) and in alternate functions of tRNA, such as initiation of reverse transcription in retroviruses (19)

References

1. F. H. C. Crick (1966) J. Mol. Biol. 19, 548–555. 

2. S. Yokoyama and S. Nishimura (1995) in tRNA: Structure, Biosynthesis, and Function (D. Söll and U. L. RajBhandary, eds.), American Society for Microbiology, Washington, DC, pp. 207223-    .

3. K. Watanabe and S. Osawa (1995) in tRNA: Structure, Biosynthesis, and Function (D. Söll and U. L. RajBhandary, eds.), American Society for Microbiology, Washington, DC, pp. 225–250. 

4. H. Ozeki, H. Inokuchi, F. K. M. Yamao, H. Sakano, T. Ikemura, and Y. Shimura (1980) in Transfer RNA: Biological Aspects (D. Söll, J. Abelson, and P. Schimmel, eds.), Cold Spring Harbor Laboratory, New York, pp. 341–362. 

5. L. Kisselev (1985) Prog. Nucleic Acid Res. Mol. Biol. 32, 237–266. 

6. L. Pallanck, M. Pak, and L.-H. Schulma (1995) in tRNA: Structure, Biosynthesis, and Function )D. Söll and U. L. RajBhandary, eds.), American Society for Microbiology, Washington DC, pp. 371–394. 

7. R. Giegé, J. D. Puglisi, and C. Florentz (1993) Prog. Nucleic Acid Res. Mol. Biol. 45, 129–206. 

8. W. H. McClain (1993) J. Mol. Biol. 234, 257–280. 

9. M. E. Saks, J. R. Sampson, and J. N. Abelson (1994) Science 263, 191–197. 

10. W. H. McClain (1995) in tRNA: Structure, Biosynthesis, and Function (D. Söll and U. L. RajBhandary, eds.), American Society for Microbiology Press, Washington, DC, pp. 335–347. 

11. J. R. Sampson, A. B. DiRenzo, L. S. Behlen, and O. C. Uhlenbeck (1989) Science 243, 13631366-    .

12. J. Putz, J. D. Puglisi, C. Florentz, and R. Giegé (1991) Science 252, 1696–1699. 

13. L. H. Schulman and H. Pelka (1988) Science 242, 765–768. 

14. S. A. Martinis and P. Schimmel (1995) in tRNA: Structure, Biosynthesis, and Function (D. Söll and U. L. RajBhandary, eds.), American Society for Microbiology Press, Washington, DC, pp. 349–370. 

15. M. Frugier, C. Florentz, and R. Giegé (1992) Proc. Natl. Acad. Sci. USA 89(9), 3990–3994. 

16. O. Nureki, T. Niimi, T. Muramatsu, H. Kanno, T. Kohno, C. Florentz, R. Giegé, and S. Yokoyama (1994) J. Mol. Biol. 236, 710–724. 

17. T. Meinnel, Y. Mechulam, S. Blanquet, and G. Fayat (1991) J. Mol. Biol. 220, 205–208. 

18. N. Mandal, D. Mangroo, J. J. Dalluge, J. A. McCloskey, and U. L. RajBhandary (1996) RNA 2, 473–482   .

19C. Isel, J.-M. Lanchy, S. F. J. Le Grice, C. Ehresmann, B. Ehresmann, and R. Marquet (1996(EMBO J. 15, 917–924.