Decoding the genome: a modified view

Abstract

Transfer RNA's role in decoding the genome is critical to the accuracy and efficiency of protein synthesis. Though modified nucleosides were identified in RNA 50 years ago, only recently has their importance to tRNA's ability to decode cognate and wobble codons become apparent. RNA modifications are ubiquitous. To date, some 100 different posttranslational modifications have been identified. Modifications of tRNA are the most extensively investigated; however, many other RNAs have modified nucleosides. The modifications that occur at the first, or wobble position, of tRNA's anticodon and those 3'-adjacent to the anticodon are of particular interest. The tRNAs most affected by individual and combinations of modifications respond to codons in mixed codon boxes where distinction of the third codon base is important for discriminating between the correct cognate or wobble codons and the incorrect near-cognate codons (e.g. AAA/G for lysine versus AAU/C asparagine). In contrast, other modifications expand wobble codon recognition, such as U*U base pairing, for tRNAs that respond to multiple codons of a 4-fold degenerate codon box (e.g. GUU/A/C/G for valine). Whether restricting codon recognition, expanding wobble, enabling translocation, or maintaining the messenger RNA, reading frame modifications appear to reduce anticodon loop dynamics to that accepted by the ribosome. Therefore, we suggest that anticodon stem and loop domain nucleoside modifications allow a limited number of tRNAs to accurately and efficiently decode the 61 amino acid codons by selectively restricting some anticodon-codon interactions and expanding others.

Figure 1

The Genetic Code. The 61 codons for the 20 amino acids and three codons for the translational stop signals are shown in the historical coding chart. Codon boxes with white backgrounds contain four codes for one amino acid and are therefore 4-fold degenerate. Codon boxes with shaded backgrounds contain codons for more than one amino acid, or an amino acid plus stop codons and therefore are 2-fold and 3-fold degenerate codons.

Figure 2

The structure and domains of tRNA. The three-dimensional structure of tRNA is represented on the left by the crystallographic structure of yeast tRNA^Phe (32). The cloverleaf secondary structure in the center is color coded to identify the structural domains of the crystal structure: amino acid accepting stem, or aminoacyl-stem (AA) is in red; dihydrouridine stem and loop domain (DSL) in black; anticodon stem and loop domain (ASL) in green; extra loop (EL) in gold; and the ribothymidine, or TΨC, stem and loop (TSL) in light blue. The positions of the invariant U33 and the amino acid accepting 3′-terminus (C74, C75 and A76) are shown. The sequence and secondary structure of the ASL from human tRNA^Lys3 is shown on the right. The nucleosides are numbered according to the accepted protocol for tRNAs, with the anticodon nucleosides as 34, 35 and 36. The modified nucleosides in ASL^Lys3 are: pseudouridine, Ψ_27,39; 5-methoxycarbonylmethyl-2-thiouridine, mcm⁵s²U₃₄; and 2-methylthio-N6-threonylcarbamoyl-adenosine, ms²t⁶A₃₇.

Figure 3

Purine 37 modifications order the anticodon loop. The structure and dynamics of triply (left) and quadruply (right) modified ASLs for yeast tRNA^Phe have been compared (142). The only difference in sequence between the two constructs is the incorporation of m¹G₃₇ versus G₃₇. Each structure is represented by a family of the 10 lowest energy structures derived from NMR and restrained molecular dynamics.

Figure 4

The Genetic Code and its decoding by tRNA. The traditional representation of the codons (left) is accompanied by tRNA’s anticodon stem and loop domain modifications known to influence decoding, translocation and/or frameshifting (Tables 1 and 2). Cognate and wobble codons that are possibly read by a wobble position, modified uridine-34 responding to one of the 4-fold degenerate codon boxes have backgrounds of white and gray. Codons representing a single amino acid in each of the mixed codon boxes are shaded in yellow and light blue. The stop codons are shaded in red. Modifications are abbreviated to represent all derivatives of the modified nucleoside, such as derivatives of xm⁵s²U and i⁶A. The wobble position 34, and purine-37 modifications (ASL right) have been discussed. Briefly, wobble position uridine modifications that include 2-thiolation (xm⁵s²U) can be required for codon binding and are restrictive to binding A and wobbling to G. The 2-thiolation promotes translocation. Modifications of the 5-carbon of uridine-34, in the absence of 2-thiolation, bind the cognate codon ending in A and promote binding to the wobble codon ending in G (xm⁵U). Oxy-modifications expand wobble beyond G to U (xo⁵U). I₃₄ binds C and wobbles to A and U. Q₃₄ promotes binding to the cognate codon ending in C over that ending in U. The position 37 modification t⁶A and its derivative ms²t⁶A promote binding to cognate codon and with position 34 uridine modifications ensure wobble codon binding and translocation. A number of position 37 modifications (i(o)⁶A, m¹G) maintain the translational reading frame.