{"title":"Sortase-Mediated Fluorescent Labeling of eIF4E for Investigating Translation Initiation Mechanisms.","authors":"Justin Pi, Simpson Joseph","doi":"10.1021/acs.biochem.4c00851","DOIUrl":null,"url":null,"abstract":"<p><p>Translation initiation represents a critical regulatory step in gene expression, orchestrated by the interaction of eukaryotic initiation factor 4E (eIF4E) with the 7-methylguanosine (m<sup>7</sup>G) cap structure at the 5' end of mRNA. This interaction enables eIF4F, composed of eIF4E, eIF4G, and eIF4A, to recruit the 43S preinitiation complex to the mRNA 5' end. The activity of eIF4E is tightly regulated and often dysregulated in cancer, neurological disorders, and viral infections. To investigate the interactions of human eIF4E with m<sup>7</sup>G-RNA and eIF4G, we engineered single-cysteine mutants of eIF4E to enable fluorescent dye attachment. However, these mutants presented challenges in purification and exhibited diminished activity. To overcome these issues, we developed a method to fluorescently label eIF4E via sortase-mediated transpeptidation. Our results demonstrate that sortase-labeled eIF4E retains activity comparable to wild-type eIF4E. This approach provides a valuable tool for studying the dynamic mechanisms of translation initiation and its regulation.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":2.9000,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biochemistry Biochemistry","FirstCategoryId":"1","ListUrlMain":"https://doi.org/10.1021/acs.biochem.4c00851","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Translation initiation represents a critical regulatory step in gene expression, orchestrated by the interaction of eukaryotic initiation factor 4E (eIF4E) with the 7-methylguanosine (m7G) cap structure at the 5' end of mRNA. This interaction enables eIF4F, composed of eIF4E, eIF4G, and eIF4A, to recruit the 43S preinitiation complex to the mRNA 5' end. The activity of eIF4E is tightly regulated and often dysregulated in cancer, neurological disorders, and viral infections. To investigate the interactions of human eIF4E with m7G-RNA and eIF4G, we engineered single-cysteine mutants of eIF4E to enable fluorescent dye attachment. However, these mutants presented challenges in purification and exhibited diminished activity. To overcome these issues, we developed a method to fluorescently label eIF4E via sortase-mediated transpeptidation. Our results demonstrate that sortase-labeled eIF4E retains activity comparable to wild-type eIF4E. This approach provides a valuable tool for studying the dynamic mechanisms of translation initiation and its regulation.
期刊介绍：
Biochemistry provides an international forum for publishing exceptional, rigorous, high-impact research across all of biological chemistry. This broad scope includes studies on the chemical, physical, mechanistic, and/or structural basis of biological or cell function, and encompasses the fields of chemical biology, synthetic biology, disease biology, cell biology, nucleic acid biology, neuroscience, structural biology, and biophysics. In addition to traditional Research Articles, Biochemistry also publishes Communications, Viewpoints, and Perspectives, as well as From the Bench articles that report new methods of particular interest to the biological chemistry community.
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