Draycheva, Albena

[["dc.bibliographiccitation.issue","1"],["dc.bibliographiccitation.journal","Scientific Reports"],["dc.bibliographiccitation.volume","12"],["dc.contributor.author","Thommen, Michael"],["dc.contributor.author","Draycheva, Albena"],["dc.contributor.author","Rodnina, Marina V."],["dc.date.accessioned","2022-09-01T09:50:07Z"],["dc.date.available","2022-09-01T09:50:07Z"],["dc.date.issued","2022"],["dc.description.abstract","Abstract\n \n Fluorescence reporter groups are important tools to study the structure and dynamics of proteins. Genetic code reprogramming allows for cotranslational incorporation of non-canonical amino acids at any desired position. However, cotranslational incorporation of bulky fluorescence reporter groups is technically challenging and usually inefficient. Here we analyze the bottlenecks for the cotranslational incorporation of NBD-, BodipyFL- and Atto520-labeled Cys-tRNA\n Cys\n into a model protein using a reconstituted in-vitro translation system. We show that the modified Cys-tRNA\n Cys\n can be rejected during decoding due to the reduced ribosome selectivity for the modified aa-tRNA and the competition with native near-cognate aminoacyl-tRNAs. Accommodation of the modified Cys-tRNA\n Cys\n in the A site of the ribosome is also impaired, but can be rescued by one or several Gly residues at the positions −1 to −4 upstream of the incorporation site. The incorporation yield depends on the steric properties of the downstream residue and decreases with the distance from the protein N-terminus to the incorporation site. In addition to the full-length translation product, we find protein fragments corresponding to the truncated N-terminal peptide and the C-terminal fragment starting with a fluorescence-labeled Cys arising from a StopGo-like event due to a defect in peptide bond formation. The results are important for understanding the reasons for inefficient cotranslational protein labeling with bulky reporter groups and for designing new approaches to improve the yield of fluorescence-labeled protein."],["dc.identifier.doi","10.1038/s41598-022-16932-7"],["dc.identifier.pii","16932"],["dc.identifier.uri","https://resolver.sub.uni-goettingen.de/purl?gro-2/113628"],["dc.language.iso","en"],["dc.notes.intern","DOI-Import GROB-597"],["dc.relation.eissn","2045-2322"],["dc.rights.uri","https://creativecommons.org/licenses/by/4.0"],["dc.title","Ribosome selectivity and nascent chain context in modulating the incorporation of fluorescent non-canonical amino acid into proteins"],["dc.type","journal_article"],["dc.type.internalPublication","yes"],["dspace.entity.type","Publication"]]

[["dc.bibliographiccitation.artnumber","5263"],["dc.bibliographiccitation.firstpage","1"],["dc.bibliographiccitation.journal","Nature Communications"],["dc.bibliographiccitation.lastpage","6"],["dc.bibliographiccitation.volume","5"],["dc.contributor.author","Ge, Yan"],["dc.contributor.author","Draycheva, Albena"],["dc.contributor.author","Bornemann, Thomas"],["dc.contributor.author","Rodnina, Marina V."],["dc.contributor.author","Wintermeyer, Wolfgang"],["dc.date.accessioned","2017-09-07T11:45:28Z"],["dc.date.available","2017-09-07T11:45:28Z"],["dc.date.issued","2014"],["dc.description.abstract","Proteins are co-translationally inserted into the bacterial plasma membrane via the SecYEG translocon by lateral release of hydrophobic transmembrane segments into the phospholipid bilayer. The trigger for lateral opening of the translocon is not known. Here we monitor lateral opening by photo-induced electron transfer (PET) between two fluorophores attached to the two SecY helices at the rim of the gate. In the resting translocon, the fluorescence is quenched, consistent with a closed conformation. Ribosome binding to the translocon diminishes PET quenching, indicating opening of the gate. The effect is larger with ribosomes exposing hydrophobic transmembrane segments and vanishes at low temperature. We propose a temperature-dependent dynamic equilibrium between closed and open conformations of the translocon that is shifted towards partially and fully open by ribosome binding and insertion of a hydrophobic peptide, respectively. The combined effects of ribosome and peptide binding allow for co-translational membrane insertion of successive transmembrane segments."],["dc.identifier.doi","10.1038/ncomms6263"],["dc.identifier.gro","3142041"],["dc.identifier.isi","000343985700002"],["dc.identifier.pmid","25314960"],["dc.identifier.uri","https://resolver.sub.uni-goettingen.de/purl?gro-2/3889"],["dc.language.iso","en"],["dc.notes.intern","WoS Import 2017-03-10"],["dc.notes.status","final"],["dc.notes.submitter","PUB_WoS_Import"],["dc.relation.issn","2041-1723"],["dc.title","Lateral opening of the bacterial translocon on ribosome binding and signal peptide insertion"],["dc.type","journal_article"],["dc.type.internalPublication","yes"],["dc.type.peerReviewed","yes"],["dc.type.subtype","original"],["dspace.entity.type","Publication"]]