It takes two to tRNAgo.

In this issue of Structure, Sievers et al.1 gain important insights into the human tRNA guanine transglycosylase QTRT1/2. The study presents a cryo-EM reconstruction of the inhibited human heterodimer in complex with human tRNAAsp, which represents the first snapshot of a eukaryotic TGT in complex with its full-length tRNA substrate.

Modulation of translational decoding by m6A modification of mRNA.

N6-methyladenosine (m6A) is an abundant, dynamic mRNA modification that regulates key steps of cellular mRNA metabolism. m6A in the mRNA coding regions inhibits translation elongation. Here, we show how m6A modulates decoding in the bacterial translation system using a combination of rapid kinetics, smFRET and single-particle cryo-EM. We show that, while the modification does not impair the initial binding of aminoacyl-tRNA to the ribosome, in the presence of m6A fewer ribosomes complete the decoding process due to the lower stability of the complexes and enhanced tRNA drop-off. The mRNA codon adopts a π-stacked codon conformation that is remodeled upon aminoacyl-tRNA binding. m6A does not exclude canonical codon-anticodon geometry, but favors alternative more dynamic conformations that are rejected by the ribosome. These results highlight how modifications outside the Watson-Crick edge can still interfere with codon-anticodon base pairing and complex recognition by the ribosome, thereby modulating the translational efficiency of modified mRNAs.

The diverse structural modes of tRNA binding and recognition.

tRNAs are short noncoding RNAs responsible for decoding mRNA codon triplets, delivering correct amino acids to the ribosome, and mediating polypeptide chain formation. Due to their key roles during translation, tRNAs have a highly conserved shape and large sets of tRNAs are present in all living organisms. Regardless of sequence variability, all tRNAs fold into a relatively rigid three-dimensional L-shaped structure. The conserved tertiary organization of canonical tRNA arises through the formation of two orthogonal helices, consisting of the acceptor and anticodon domains. Both elements fold independently to stabilize the overall structure of tRNAs through intramolecular interactions between the D- and T-arm. During tRNA maturation, different modifying enzymes posttranscriptionally attach chemical groups to specific nucleotides, which not only affect translation elongation rates but also restrict local folding processes and confer local flexibility when required. The characteristic structural features of tRNAs are also employed by various maturation factors and modification enzymes to assure the selection, recognition, and positioning of specific sites within the substrate tRNAs. The cellular functional repertoire of tRNAs continues to extend well beyond their role in translation, partly, due to the expanding pool of tRNA-derived fragments. Here, we aim to summarize the most recent developments in the field to understand how three-dimensional structure affects the canonical and noncanonical functions of tRNA.

Rhodopsin-bestrophin fusion proteins from unicellular algae form gigantic pentameric ion channels.

Many organisms sense light using rhodopsins, photoreceptive proteins containing a retinal chromophore. Here we report the discovery, structure and biophysical characterization of bestrhodopsins, a microbial rhodopsin subfamily from marine unicellular algae, in which one rhodopsin domain of eight transmembrane helices or, more often, two such domains in tandem, are C-terminally fused to a bestrophin channel. Cryo-EM analysis of a rhodopsin-rhodopsin-bestrophin fusion revealed that it forms a pentameric megacomplex (~700 kDa) with five rhodopsin pseudodimers surrounding the channel in the center. Bestrhodopsins are metastable and undergo photoconversion between red- and green-absorbing or green- and UVA-absorbing forms in the different variants. The retinal chromophore, in a unique binding pocket, photoisomerizes from all-trans to 11-cis form. Heterologously expressed bestrhodopsin behaves as a light-modulated anion channel.