The Modified Wobble Base Inosine in Yeast tRNA^Ile Is a Positive Determinant for Aminoacylation by Isoleucyl-tRNA Synthetase

Abstract

Earlier work by two independent groups has established the fact that anticodons GAU and LAU of Escherichia coli tRNAIle isoacceptors play a critical role in the tRNA identity. Yeast possesses two isoleucine transfer RNAs, a major one with anticodon IAU and a minor one with anticodon PsiAPsi which are derived from the post-transcriptional modification of AAU and UAU gene sequences, respectively. We present direct evidence which reveals that inosine is a positive determinant for yeast isoleucyl-tRNA synthetase. We also show that yeast tRNAMet with guanosine at the wobble position becomes aminoacylated with isoleucine while methionine acceptance is lost. As inosine and guanosine share the 6-keto and the N-1 hydrogen groups, this suggests that these hydrogen donor and acceptor groups are determinants for isoleucine specificity. The role of the minor tRNAIle anticodon pseudouridines in tRNA isoleucylation could not be tested directly but was deduced from a 40-fold decrease in the activity of the unmodified transcript. The presence of the NHCO structure in guanosine, inosine, pseudouridine, and lysidine suggests a unifying model of wobble base recognition by the yeast and E. coli isoleucyl-tRNA synthetase. In contrast to lysidine which switches the identity of the tRNA from methionine to isoleucine [Muramatsu, T., Nishikawa, K., Nemoto, F., Kuchino, Y., Nishimura, S., Miyazawa, T., & Yokoyama, S. (1988) Nature 336, 179-181], pseudouridine-34 does not modify the specificity of the yeast minor tRNAIle since U-34 is a strong negative determinant for yeast MetRS. Therefore, the major role of Psi-34 (in combination with Psi-36 or not) is likely in isoleucine AUA codon specificity and translational fidelity.

Redundant Synthesis of Cysteinyl-tRNACys in Methanosarcina mazei

“…It appears quite possible, therefore, that further base or ribose modification may be required for optimal Cys-tRNA Cys formation by SepRS, CysRS, or both enzymes. A positive tRNA modification requirement for efficient aminoacylation has been observed previously in only a small number of cases, primarily involving the wobble nucleotide at the 5Ј-position of the anticodon in the isoleucyl, glutamyl, and glutaminyl systems (27)(28)(29)(30)(31). The only previous observation of a positive tRNA methylation requirement for aminoacylation is the need for 1-methyladenosine at position 9 of nematode mitochondrial tRNAs lacking the T-arm (32).…”

Pseudouridine in RNA: What, Where, How, and Why

“…Also, eukaryotic cytoplasmic tRNA Tyr (anticodon GW A) is a suppressor of UAA and UAG stop codons (see 48). In yeast, a minor tRNA Ile variant has the anticodon W AW (49). In these contexts, W 34, W 35, and W 36 are postulated to strengthen codon-anticodon base-pairing, particularly noncanonical interactions involving G-A or G-G wobble base pairs (11).…”

Celebrating wobble decoding: Half a century and still much is new

“…118-120 The C14 alcohol of ct 6 A can hydrogen bond with the N7 of the first codon, an A, increasing the stabilization that enhanced stacking alone provides. 118,120,121 …”

“…The modification lysidine is the recognition determinant for the amino acid specificity in isoleucyl-tRNA synthetase (IleRS) aminoacylation of tRNA Ile LAU and not tRNA Met CAU . An intriguing minor tRNA Ile ΨAΨ in yeast contains pseudouridine at position 34 instead of lysidine, 121 and many archeal species utilize agmatidine [N-(4-carbamimidamidobutyl)-4-imino-1-(b-D-ribofuranosyl)-1,4-dihydro-2-pyrimidinamine] (Fig. 3B) for decoding of the AUA isoleucine codon.…”

“…122,126 The hydrogen bonding capability of modified nucleosides commonly found at the wobble position of tRNA Ile illustrates how small changes in the chemistry of a single nucleoside can have broad and highly specific effects, in this case altering codon recognition and specificity while simultaneously preserving aaRS recognition. 121 …”