title: Trigger Factor-Induced Nascent Chain Dynamics Changes Suggest Two Different Chaperone-Nascent Chain Interactions during Translation.
abstract: Protein biogenesis is poorly understood due to the ribosome that perturbs measurement attempted on the ribosome-bound nascent chain (RNC). Investigating nascent chain dynamics may provide invaluable insight into the co-translational processes such as structure formation or interaction with a chaperone [e.g., the bacterial trigger factor (TF)]. In this study, we aim to establish a platform for studying nascent chain dynamics by exploring the local environment near the fluorescent dye on site-specifically labeled RNCs with time-resolved fluorescence anisotropy. To prepare a quantitative model of fluorescence depolarization, we utilized intrinsically disordered protein bound to ribosome, which helped us couple the sub-nanosecond depolarization with the motion of the nascent chain backbone. This was consistent with zinc-finger-domain-containing RNCs, where the extent of sub-nanosecond motion decreased upon the addition of zinc when the fluorophore was in close proximity of the domain. After the characterization of disordered nascent chain dynamics, we investigated the synthesis of a model cytosolic protein, Entner-Doudoroff aldolase, labeled at different sites during various stages of translation. Depending on the stage of translation, the addition of the TF to the nascent chain led to two different responses in the nascent chain dynamics serendipitously, suggesting steric hindrance between the nascent chain and the chaperone as a mechanism for TF dissociation from the ribosome during translation. Overall, our study demonstrates the possible use of site-specific labeling and time-resolved anisotropy to gain insight on chaperone binding event at various stages of translation and hints on TF co-translational mechanism.
authors.affiliation: Institute of Chemistry, Academia Sinica, Taipei, Taiwan, 11529, R.O.C.
authors.forename: Jiří
authors.initials: J
authors.lastname: Koubek
authors.affiliation: Institute of Chemistry, Academia Sinica, Taipei, Taiwan, 11529, R.O.C.
authors.forename: Yi-Che
authors.initials: YC
authors.lastname: Chang
authors.affiliation: Institute of Chemistry, Academia Sinica, Taipei, Taiwan, 11529, R.O.C.
authors.forename: Sunny Yao-Chen
authors.initials: SY
authors.lastname: Yang
authors.affiliation: Institute of Chemistry, Academia Sinica, Taipei, Taiwan, 11529, R.O.C.. Electronic address: jthuang@gate.sinica.edu.tw.
authors.forename: Joseph Jen-Tse
authors.initials: JJ
authors.lastname: Huang
mesh_terms: D029968:Escherichia coli Proteins / Q000378:metabolism*; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry* / Q000378:metabolism*; D019696:Peptidylprolyl Isomerase / Q000378:metabolism*; D014176:Protein Biosynthesis; D017510:Protein Folding
chemical_list: D029968:Escherichia coli Proteins; D005634:Fructose-Bisphosphate Aldolase; C403324:trigger factor, E coli; D019696:Peptidylprolyl Isomerase
keywords: chaperone–nascent chain interaction; co-translational protein folding; ribosome-bound nascent chain; site-specific labeling; time-resolved fluorescence anisotropy