{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T04:55:39Z","timestamp":1790916939156,"version":"4.1.0"},"reference-count":22,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T00:00:00Z","timestamp":1790899200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Mol. Biosci."],"abstract":"<jats:p>\n                    Translation termination in eukaryotes requires the concerted action of release factors eRF1 and eRF3, together with accessory factors including eIF5A and Rli1, to achieve efficient peptide release from the ribosome. Here, we establish a yPURE system-based Termi-nLuc assay, in which pre-termination complexes (PreTCs), carrying full-length nanoluciferase (nLuc) peptidyl-tRNA, are formed by simply omitting release factors from the reconstituted yeast translation system, enabling rapid and sensitive analysis without the need for sucrose density gradient purification. To enable direct biochemical analysis of GGQ methylation\u2014a physiologically important modification of eRF1 whose effects on peptide release activity had not previously been examined\n                    <jats:italic>in vitro<\/jats:italic>\n                    \u2014we also established an\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    co-expression system of eRF1 and its GGQ-methylating enzyme, yielding fully GGQ-methylated eRF1. Using this platform, we characterized the individual and combined contributions of eRF1, eRF3, eIF5A, and Rli1 to peptide release, and demonstrated that the full complement of these factors is sufficient for efficient release. Under the current assay conditions, neither GGQ methylation of eRF1 nor the identity of the penultimate amino acid of the protein detectably affected peptide release activity, consistent with the possibility that eRF1\u2022eRF3 recruitment to the ribosome, rather than the chemical hydrolysis step, is rate-limiting under these conditions. The yPURE-based Termi-nLuc assay is readily amenable to manipulation of the composition of translation factors, tRNAs, and mRNA sequences, making it a versatile and high-throughput-compatible platform for future studies of context-dependent regulation of translation termination, with potential applications in the screening of small molecules that modulate termination efficiency or promote readthrough of premature stop codons for the treatment of genetic diseases.\n                  <\/jats:p>","DOI":"10.3389\/fmolb.2026.1933983","type":"journal-article","created":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T04:40:53Z","timestamp":1790916053000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Full-protein release assay in a reconstituted yeast translation system using GGQ-methylated eRF1"],"prefix":"10.3389","volume":"13","author":[{"given":"Shuai","family":"Huang","sequence":"first","affiliation":[{"name":"Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo","place":["Chiba, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yichen","family":"Xu","sequence":"additional","affiliation":[{"name":"Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo","place":["Chiba, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenting","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo","place":["Chiba, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ziang","family":"Miao","sequence":"additional","affiliation":[{"name":"Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo","place":["Chiba, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chang","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo","place":["Chiba, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Riku","family":"Nagai","sequence":"additional","affiliation":[{"name":"Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo","place":["Chiba, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Muhoon","family":"Lee","sequence":"additional","affiliation":[{"name":"Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo","place":["Chiba, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hitomi","family":"Seike","sequence":"additional","affiliation":[{"name":"Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo","place":["Chiba, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shinji","family":"Nagata","sequence":"additional","affiliation":[{"name":"Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo","place":["Chiba, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nono","family":"Takeuchi-Tomita","sequence":"additional","affiliation":[{"name":"Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo","place":["Chiba, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,10,2]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"441","DOI":"10.1093\/jb\/mvaa021","article-title":"Reconstitution of yeast translation elongation and termination in vitro utilizing CrPV IRES-containing mRNA","volume":"167","author":"Abe","year":"","journal-title":"J. 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