Acceptor Stem Differences Contribute to Species-Specific Use of Yeast and Human tRNASer

Abstract

The molecular mechanisms of translation are highly conserved in all organisms indicative of a single evolutionary origin. This includes the molecular interactions of tRNAs with their cognate aminoacyl-tRNA synthetase, which must be precise to ensure the specificity of the process. For many tRNAs, the anticodon is a major component of the specificity. This is not the case for the aminoacylation of alanine and serine to their cognate tRNAs. Rather, aminoacylation relies on other features of the tRNA. For tRNASer, a key specificity feature is the variable arm, which is positioned between the anticodon arm and the T-arm. The variable arm is conserved from yeast to human. This work was initiated to determine if the structure/function of tRNASer has been conserved from Saccharomyces cerevisiae to human. We did this by detecting mistranslation in yeast cells with tRNASer derivatives having the UGA anticodon converted to UGG for proline. Despite being nearly identical in everything except the acceptor stem, human tRNASer is less active than yeast tRNASer. A chimeric tRNA with the human acceptor stem and other sequences from the yeast molecule acts similarly to the human tRNASer. The 3:70 base pair in the acceptor stem (C:G in yeast and A:U in humans) is a prime determinant of the specificity. Consistent with the functional difference of yeast and human tRNASer resulting from subtle changes in the specificity of their respective SerRS enzymes, the functionality of the human and chimeric tRNASerUGG molecules was enhanced when human SerRS was introduced into yeast. Residues in motif 2 of the aminoacylation domain of SerRS likely participated in the species-specific differences. Trp290 in yeast SerRS (Arg313 in humans) found in motif 2 is proximal to base 70 in models of the tRNA-synthetase interaction. Altering this motif 2 sequence of hSerRS to the yeast sequence decreases the activity of the human enzyme with human tRNASer, supporting the coadaptation of motif 2 loop–acceptor stem interactions.

2'-O-ribose methylation of transfer RNA promotes recovery from oxidative stress in Saccharomyces cerevisiae

“…Another possibility is that Trm3-mediated position 4 methylation influences other processes of translation, such as translocation or aminoacylation. Indeed, in E. coli, 2'-O-ribose methylation within the acceptor stem can negatively influence translation [47], and some aminoacyl tRNA synthetases rely on tRNA features within the acceptor stem or variable loop region to determine tRNA identity for charging [48][49][50][51].…”

“…Um44 modification is thought to increase tRNA stability by maximizing base stacking and base interactions that promote the C3'-endo form (preferred for stability in thermophiles) of RNA [55]. Another role for Trm44-mediated Um44 modification could be linked to charging of tRNA Ser by seryl-tRNA synthetases, which rely on features within the acceptor stem and variable loop regions for recognition [48][49][50][51]. Thus, both stability and accurate tRNA Ser charging could be affected in trm44 mutants, producing sensitivity to oxidative stress.…”

Synthetic DNA and RNA Programming

“…Although Crick’s frozen accident hypothesis once envisioned a perfectly interpreted genetic code [19], recent studies have found that cells tolerate surprisingly high levels of amino acid misincorporation [20]. The next pair of featured articles [1,2] investigated biochemical mechanisms that regulate translation fidelity.…”

“…Berg et al [1] provided the first detailed characterization of human tRNA Ser identity elements. These are the critical nucleotides that are essential for tRNA recognition by the cognate aminoacyl-tRNA synthetase and determine the serine accepting identity of this tRNA.…”

“…The approaches highlighted here involve programming genes, RNAs, and proteins, both in the test tube and in living cells, to explore areas of molecular biology related to genetic code evolution [1,2] and genetic code expansion [3,4,5], including applications in programming protein modifications [6,7], RNA metabolism [8,9], and genetic systems for genome engineering [10,11] and biocontainment of modified microorganisms [12]. These contributions showcase both the diversity of research approaches emerging as synthetic biology tools and the extreme level of detail with which these tools enable studies and the manipulation of individual protein modifications or cellular pathways.…”