Biology subjects

Wakabayashi, H.

Publications and source records attributed to Wakabayashi, H..

4 recordsLinked to original sources

bioRxiv2025

Observing intersubunit dynamics in single yeast ribosomes

Translation is accompanied by the rotation of the small and large ribosomal subunits relative to each other. Here, we use single-molecule Forster resonance energy transfer between fluorophores introduced into ribosomal proteins uS15 and eL30 to follow the intersubunit dynamics of Saccharomyces cerevisiae ribosomes. Similar to their bacterial counterparts, yeast ribosomes are observed to sample two predominant FRET states corresponding to the nonrotated (NR) and rotated (R) conformations. Our data yield further evidence that intersubunit rotation is coupled to tRNA transitions between the classical and hybrid binding states. In particular, the transition from the NR to R conformation is inhibited by the antibiotic cycloheximide, which is known to bind to the E site of the large subunit and prevent the movement of deacylated tRNA into the hybrid P/E state. The elongation cycle, which comprises tRNA binding, peptide transfer, and mRNA-tRNA translocation, is accompanied by switching from NR to R, and then back to the NR conformation. We find that fungal elongation factor 3 (eEF3) stabilizes the NR conformation of the ribosome. Our data are consistent with the model suggesting that eEF3 facilitates E-site tRNA release at the late step of mRNA-tRNA translocation, following the reverse intersubunit rotation induced by the universally conserved elongation factor 2 (eEF2). Our uS15-eL30 smFRET assay provides the basis for investigating eukaryotic mechanisms of translation regulation, including ribosome pausing, stalling, and frameshifting.

biophysics↗

bioRxiv2025

40S ribosomal subunits scan mRNA for the start codon by one-dimensional diffusion

During eukaryotic translation initiation, the small (40S) ribosomal subunit is recruited to the 5' cap and subsequently scans the 5' untranslated region (5' UTR) of mRNA in search of the start codon. The molecular mechanism of mRNA scanning remains unclear. Here, using GFP reporters in Saccharomyces cerevisiae cells, we show that order-of-magnitude variations in the lengths of unstructured 5' UTRs have a modest effect on protein synthesis. These observations indicate that mRNA scanning is not rate limiting in yeast cells. Conversely, the presence of secondary structures in the 5' UTR strongly inhibits translation. Loss-of-function mutations in translational RNA helicases eIF4A and Ded1, as well as mutations in other initiation factors implicated in mRNA scanning, namely eIF4G, eIF4B, eIF3g and eIF3i, produced a similar decrease in translation of GFP reporters with short and long unstructured 5' UTRs. As expected, mutations in Ded1, eIF4B and eIF3i severely diminished translation of the reporters with structured 5' UTRs. Evidently, while RNA helicases eIF4A and Ded1 facilitate 40S recruitment and secondary structure unwinding, they are not rate-limiting for the 40S movement along the 5' UTR. Hence, our data indicate that, instead of helicase-driven translocation, one-dimensional diffusion predominately drives mRNA scanning by the 40S subunits in yeast cells.

genetics↗

bioRxiv2021

Specific length and structure rather than high thermodynamic stability enable regulatory mRNA stem-loops to pause translation

Translating ribosomes unwind mRNA secondary structures by three basepairs each elongation cycle. Despite the ribosome helicase, certain mRNA stem-loops stimulate programmed ribosomal frameshift by inhibiting translation elongation. Here, using mutagenesis, biochemical and single-molecule experiments, we examine whether high stability of three basepairs, which are unwound by the translating ribosome, is critical for inducing ribosome pauses. We find that encountering frameshift-inducing mRNA stem-loops from the E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) hinders A-site tRNA binding and slows down ribosome translocation by 15-20 folds. By contrast, unwinding of first three basepairs adjacent to the mRNA entry channel slows down the translating ribosome by only 2-3 folds. Rather than high thermodynamic stability, specific length and structure enable regulatory mRNA stem-loops to stall translation by forming inhibitory interactions with the ribosome. Our data provide the basis for rationalizing transcriptome-wide studies of translation and searching for novel regulatory mRNA stem-loops.

biochemistry↗

bioRxiv2020

Extending the spacing between the Shine-Dalgarno sequence and P-site codon reduces the rate of mRNA translocation

By forming basepairing interactions with the 3 end of 16S rRNA, mRNA Shine-Dalgarno (SD) sequences positioned upstream of Open Reading Frames (ORFs) facilitate translation initiation. During the elongation phase of protein synthesis, intragenic SD-like sequences stimulate ribosome frameshifting and may also slow down ribosome movement along mRNA. Here, we show that the length of the spacer between the SD sequence and P-site codon strongly affects the rate of ribosome translocation. Increasing the spacer length beyond six nucleotides destabilizes mRNA-ribosome interactions and results in a 5-10 fold reduction of the translocation rate. These observations suggest that during translation, the spacer between the SD sequence and P-site codon undergoes structural rearrangements, which slow down mRNA translocation and promote mRNA frameshifting.

biochemistry↗