| During ribosome-mediated protein synthesis, non-initiator tRNAs act as translating chaperones by transferring the correct amino acid to the ribosome through sequence-specific interactions with mRNA codons in the ribosomal A-site. To achieve uniformity in codon recognition ability between different tRNAs, the anticodon domains of all must adopt a singular conformation in the ribosomal decoding center. Since there is a necessary sequence variation for the three anticodon residues, innovative ways of attaining a constant loop structure are required. Nature has resolved this issue by introducing functional groups to the loop residues, changing the chemical environment and, thus, the conformation. In fact, there is a large diversity and number of these modifications found in tRNAs of all known life.;Escherichia coli (E. coli) arginyl-tRNAs offer the opportunity to study these modifications in the context of six-fold degenerate codons that vary widely in their cellular usage. A single set of very similar isoacceptors, tRNAArg1ICG and tRNA Arg2ICG, have anticodon domains that differ only in the presence or absence of a very rare modification, 2-thiocytidine (s 2C32). These tRNAs are particularly interesting not only because of the rare s2C32 modification, but also through the wobble position modification I34, they are both responsible for decoding two very common codons CGU and CGC as well as the very rare CGA codon. Typically, the codon usage correlates with the tRNA content of the cell; however, in this instance, the tRNA content is high which does not match what would be expected for an isoacceptor that recognizes a rare codon. Here we have shown conclusively that modifications can act to restrict the decoding capability of the tRNA to only the two common codons, suggesting that the tRNA is present at high levels, consistent with decoding common codons, but that there is a smaller pool of tRNA in different states of modification, corresponding to decoding of the rare codon. The mechanism of this function is not completely understood, however, we show that the modifications do not accomplish this through a completely structural device. In this case, modifications act to modulate the flexibility of specific loop residues, thereby modulating the conformational landscape that must be traversed between two disparate, but necessary, cellular conformations.;It is clear from many previous studies that modifications can perform specific functions in the anticodon stem and loop (ASL), most notably at position 34 which is known to facilitate the "wobble" recognition of multiple codons. E. coli tRNAArg4UCU offers a novel function of modification at this position that is not fully understood. In a very similar isoacceptor, partially modified tRNALys UUU, a very different decoding function is seen. Both of these tRNAs contain the same loop sequence with the exception that tRNAArg4 UCU has a C at position 35, whereas tRNALysUUU has U35. Interestingly, they both contain the same loop modifications, mnm5U34 and t6A37, with the exception that tRNAArg4UCU has an additional s2C32 modification; however tRNALys UUU, in this particular modification state can decode both AAA and AAG and tRNAArg4UCU decodes only AGA. Although s 2C32 enhances codon discrimination in tRNAArg1 ICG, codon-specific ribosome binding assays clearly refute this role in tRNAArg4UCU. The modifications, however, appear to impart similar biophysical and thermodynamic properties to the ASL as in tRNALysUUU, therefore a full structural characterization is underway using NMR spectroscopy to ascertain whether modifications drive the conformation toward a canonical U-turn conformation as with tRNA LysUUU. Preliminary evidence indicates a role for modifications in altering the stability of the non-canonical C32•A 38 base pair through stable stacking interactions and elevation of A 38H1 pKa. |
