title: Functional dissection of Escherichia coli trigger factor: unraveling the function of individual domains.
abstract: In Escherichia coli, the ribosome-associated chaperone Trigger Factor (TF) promotes the folding of newly synthesized cytosolic proteins. TF is composed of three domains: an N-terminal domain (N), which mediates ribosome binding; a central domain (P), which has peptidyl-prolyl cis/trans isomerase activity and is involved in substrate binding in vitro; and a C-terminal domain (C) with unknown function. We investigated the contributions of individual domains (N, P, and C) or domain combinations (NP, PC, and NC) to the chaperone activity of TF in vivo and in vitro. All fragments comprising the N domain (N, NP, NC) complemented the synthetic lethality of Deltatig DeltadnaK in cells lacking TF and DnaK, prevented protein aggregation in these cells, and cross-linked to nascent polypeptides in vitro. However, DeltatigDeltadnaK cells expressing the N domain alone grew more slowly and showed less viability than DeltatigDeltadnaK cells synthesizing either NP, NC, or full-length TF, indicating beneficial contributions of the P and C domains to TF's chaperone activity. In an in vitro system with purified components, none of the TF fragments assisted the refolding of denatured d-glyceraldehyde-3-phosphate dehydrogenase in a manner comparable to that of wild-type TF, suggesting that the observed chaperone activity of TF fragments in vivo is dependent on their localization at the ribosome. These results indicate that the N domain, in addition to its function to promote binding to the ribosome, has a chaperone activity per se and is sufficient to substitute for TF in vivo.
authors.affiliation: Zentrum für Molekulare Biologie (ZMBH), Universität Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany.
authors.forename: G
authors.initials: G
authors.lastname: Kramer
authors.forename: A
authors.initials: A
authors.lastname: Rutkowska
authors.forename: R D
authors.initials: RD
authors.lastname: Wegrzyn
authors.forename: H
authors.initials: H
authors.lastname: Patzelt
authors.forename: T A
authors.initials: TA
authors.lastname: Kurz
authors.forename: F
authors.initials: F
authors.lastname: Merz
authors.forename: T
authors.initials: T
authors.lastname: Rauch
authors.forename: S
authors.initials: S
authors.lastname: Vorderwülbecke
authors.forename: E
authors.initials: E
authors.lastname: Deuerling
authors.forename: B
authors.initials: B
authors.lastname: Bukau
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism*; D009154:Mutation; D019696:Peptidylprolyl Isomerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D012270:Ribosomes / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; C063842:dnaK protein, E coli; C403324:trigger factor, E coli; D019696:Peptidylprolyl Isomerase