Nagao, A. | Search bioRxiv

bioRxiv2024

Structural insight into bacterial co-transcriptional translation initiation

In bacteria, transcription and translation are tightly coupled, forming a transcription-translation complex (TTC) between RNA polymerase (RNAP) and the ribosome. As nascent mRNA emerging from RNAP is susceptible to ribonuclease digestion, undesired RNA folding, and R-loop formation, immediate TTC formation is important. Here, we report the cryo-electron microscopy structures that capture the translation initiation complex assembly on transcribing RNAP. As a short mRNA emerging from RNAP, the 30S ribosomal subunit binds the RNAP on its inter-subunit side, interacting with the mRNA 5-region and the initiator tRNA. The RNAP could relocate to the canonical mRNA-entry site of 30S, threading the mRNA into a path formed between RNAP and 30S. The subsequent 50S joining establishes a TTC. These structures illustrate the transcription-coupled translation initiation, while protecting mRNA. One-Sentence SummaryCryo-EM captures the ribosome assembly on transcribing RNA polymerase in the presence of translation initiation factors.

biochemistry↗

bioRxiv2023

Complexity and dynamics of in organello translation landscape assessed by high-resolution mitochondrial ribosome profiling

Since mitochondrial translation serves the essential subunits of the OXPHOS complex that produces ATP, exhaustive, quantitative, and high-resolution delineation of mitoribosome traversal is needed. Here, we developed a technique for high-resolution mitochondrial ribosome profiling and revealed the intricate regulation of mammals in organello translation. Our approach assessed the stoichiometry and kinetics of mitochondrial translation flux, such as the number of mitoribosomes on a transcript and the elongation rate, initiation rate, and lifetime rounds of translation of individual transcripts. We also surveyed the impacts of modifications at the anticodon stem loop in mt-tRNAs, including all possible modifications at the 34th position, by deleting the corresponding enzymes and harnessing patient-derived cells. Moreover, a retapamulin-assisted derivative and mito-disome profiling revealed cryptic translation initiation sites at subcognate codons and programmed mitoribosome collision sites across the mitochondrial transcriptome. Our work provides a useful platform for investigating protein synthesis within the energy powerhouse of the cell.

molecular biology↗