Tuning tRNAs for improved translation

Abstract

Transfer RNAs have been extensively explored as the molecules that translate the genetic code into proteins. At this interface of genetics and biochemistry, tRNAs direct the efficiency of every major step of translation by interacting with a multitude of binding partners. However, due to the variability of tRNA sequences and the abundance of diverse post-transcriptional modifications, a guidebook linking tRNA sequences to specific translational outcomes has yet to be elucidated. Here, we review substantial efforts that have collectively uncovered tRNA engineering principles that can be used as a guide for the tuning of translation fidelity. These principles have allowed for the development of basic research, expansion of the genetic code with non-canonical amino acids, and tRNA therapeutics.

Keywords: directed evolution; genetic code expansion; noncanonical amino acid; rational design; synthetic biology; tRNA engineering; tRNA therapeutics; translation.

FIGURE 1

Schematic of tRNA structure. tRNAs consist of two major domains (acceptor domain and anticodon domain) and five stem-loop arms (acceptor arm (blue), D-arm (green), anticodon arm (pink and white), variable arm (orange), and T-arm (gold)) which play different roles in translation. (A) Cloverleaf 2D structure of tRNA^Asp (PDB: 6UGG) highlighting the major domains and regions. (B) 3D stick representation of tRNA^Asp with domains highlighted.

FIGURE 2

Overview of translation initiation and elongation highlighting important tRNA interactions. During translation, tRNA frequently interacts with both the ribosome and a host of translation factors, playing a central role in all major translational processes. (A) Initiation factors (gold, black, and red) help guide the initiator aa-tRNA into the ribosomal P-site to form the 30S initiation complex. Then, the initiation factors release and allow another aa-tRNA to accommodate into the A-site which catalyzes the first peptide bond to form, marking the beginning of elongation. (B) In translation elongation, the aa-tRNA must bind EF-Tu/eEF1A to be transported to the ribosomal A/T site. Codon-anticodon interactions between the aa-tRNA, mRNA, and ribosome ensure the correct aa-tRNA is selected before peptidyl transfer between the P-site and A-site tRNAs occur. Additionally, the P-site tRNA binds EF-P/eIF5A when tRNAs are charged with difficult ribosomal substrates to improve peptidyl transfer efficiency. Finally, EF-G/eEF2 interacts with the ribosome and A-site aa-tRNA to catalyze translocation. Created with BioRender.com.

FIGURE 3

tRNA interacts with EF-Tu to moderate ribosome acceptance of aa-tRNAs. (A) Visualization of E. coli Cys-tRNA^Cys (blue) bound to EF-Tu (orange) (PDB: 1B23). The black box highlights EF-Tu interactions with the acceptor domain of the tRNA. A zoom-in on the box shows the specific bases involved (highlighted in bright green). Base pair 7:66 (olive green) contributes least to the affinity, while the other three pairs contribute more. (B) Graph showing the different binding affinities of natural elongator valine-charged E. coli tRNAs to EF-Tu (Asahar and Uhlenbeck, 2002: Copyright (2002) National Academy of Science, United.States). (C) Different T-stem base pairs [highlighted in panels (A, B)] modularly impact aa-tRNA binding to EF-Tu. The bolded base pairs for each of the three T-stem base pairs represent the natural sequence when calculating the relative ΔΔG° to compare binding affinity differences. The intensity of red and blue shading indicates a base pair with relatively higher or lower affinity to EF-Tu at that position, respectively. Data from Schrader and Uhlenbeck, (2011) with E. coli tRNA^Phe and figure repurposed from Shrader and Uhlenbeck, (2018).

FIGURE 4

tRNA engineering sites explored to improve translational fidelity. (A) Bacterial tRNA regions have shown utility in improving various aspects of translation, such as ribosome acceptance (gold), decoding (pink), peptidyl transfer (green), translocation (orange), and tertiary structure (blue). (B) Eukaryotic tRNA regions engineered to improve their translational fidelity. Directed evolution experiments have elucidated the acceptor stem and anticodon stem as sites that non-specifically improve eukaryotic tRNA translational fidelity (grey), and other regions have been rationally engineered similarly to bacterial systems.

FIGURE 5

tRNA sites explored in quadruplet tRNAs to improve translational fidelity. Efficient quadruplet tRNAs have a well-conserved G:C/C:G base pair adjacent to the anticodon loop (yellow), A37 and U33 residues, and base pair mismatches near the beginning of the anticodon stem (blue) which adjusts the conformation of tRNAs. Frequently post-transcriptionally modified residues (purple boxes) also impact translational fidelity, although position 33.5 base pairing with a quadruplet codon is not necessary.

FIGURE 6

Initiator tRNA engineering sites. Initiator tRNAs contain unique sequence features (light blue) to encourage proper aminoacylation and binding to the ribosome P-site, as opposed to elongator tRNAs which bind the A-site. Sequences governing EF-Tu (green) and EF-P (orange) affinity have been carried over from elongator tRNA engineering research to improve initiator tRNA fidelity. Additionally, directed evolution has determined some non-specific improvements (grey) in the anticodon stem that further improve fidelity, including a G51-G52 motif in the T-stem that enhances IF2 binding to help position initiator tRNAs in the ribosomal P-site.