Structural and chemical features for accurate reading of the genetic code by the ribosome and tRNA (renewal)

In all living organisms, the genetic information carried by the messenger RNA (mRNA) is translated into proteins by the ribosome. Faithful decoding of the genes is central to cell homeostasis. The shape and chemical complementarity of the ribosome and tRNAs seamlessly go hand-in-hand to perform this elaborated task. During reading of the genetic code, the ribosome monitors the quality of the base pairs between mRNA codons and tRNAs. This is accomplished by conformational changes in the ribosome, tRNAs, and translation factors. To achieve high accuracy, the genetic sequence of tRNA is not enough; the cell relies on the epitranscriptomic of RNA. Recently, modification of mRNA by the incorporation of pseudouridines has proven to be critical for efficient translation of the spike protein of SARS-CoV-2, the main component of the mRNA-based vaccines against COVID19. The cellular epitranscriptome does not only include mRNA and rRNA, but also tRNA molecules. The field of RNA modification is currently expending exponentially with the potential to lead to novel therapeutics. We are only beginning to assess the effects of the mRNA epitranscriptome on translation efficiency and accuracy. In this project, we leverage our expertise as structural biologists investigating protein synthesis to understand at the molecular level how modifications of nucleotides in tRNA and mRNA regulate translation of the genetic code. tRNA molecules contain the highest concentration of modified nucleotides, which are shown to facilitate folding of tRNAs, recognition by aminoacyl-tRNA synthetases, maintenance of the reading frame, and accuracy of mRNA decoding. Accurate decoding is not only driven by the formation of Watson-Crick base pairs between mRNA and the anticodon loop of tRNA, but also by restricting recognition of near-cognate codons. While the first and second positions of the codon are strictly monitored by the ribosome decoding center in the small (30S) subunit, allowing essentially only Watson-Crick or Watson-Crick-like geometry, the third position of the codon is not as closely monitored by the ribosome. The third, or “wobble”, position therefore allows for the formation of non-canonical base pairs, which accounts for the degeneracy of the genetic code. This permits one tRNA to bind to multiple codons directing the incorporation of a single amino acid. Therefore, it is particularly challenging for tRNAs and the ribosome to discriminate codons solely from the nucleotide identity at the third position of mRNA. To circumvent this fundamental issue, the cell uses post-transcriptional modifications of tRNAs. Modifications of nucleotide 34 (wobble position) in the anticodon loop of tRNAs can expand or restrict the decoding capacity. The presence of uridine 5-oxyacetic acid at the wobble position of tRNA allows tRNAUACVal, tRNAUGCAla, and tRNAUGGPro to pair with adenosine, guanosine, uridine, and cytosine at the third position of mRNA . Similarly, deamination of adenosine 34 to inosine allows it to pair with U, C, and A . Conversely, there are two examples in the genetic code where the wobble position of tRNA should discriminate between G and A in mRNA. The first one occurs between the UGG (Trp) and UGA (stop) codons which is, however, accompanied with a high level of UGA readthrough , illustrating the challenge of discrimination between purines at the wobble position of mRNA. In the second example, the minor tRNACAUIle decodes specifically its cognate AUA codon, while avoiding the AUG (Met) codon. For this, C34 is modified to lysidine (k2C) by the addition of a lysine residue at position C2 of cytosine 34 by the enzyme TilS (see preliminary data). Similarly, pioneer studies reported that the elongator tRNAMet (tRNAeMet) with the CAU anticodon requires modification of C34 through the addition of an acyl group onto position N4 of cytosine (ac4C). This also increases the accuracy of decoding because unmodified tRNAeMet was shown to cross-read the AUA codon (see Aim 1). Recently, the enzyme that performs this modification was identified in Escherichia coli and Bacillus subtillis. The overarching goal of this project is to elucidate how the epitranscriptome of tRNA and mRNA, combined with specific structural features in the ribosome, translation factors, and tRNA itself, accomplish the most fundamental task of translating the genetic code into functional proteins.