title: Conformational changes in switch I of EF-G drive its directional cycling on and off the ribosome.
abstract: We have trapped elongation factor G (EF-G) from Escherichia coli in six, functionally defined states, representing intermediates in its unidirectional catalytic cycle, which couples GTP hydrolysis to tRNA-mRNA translocation in the ribosome. By probing EF-G with trypsin in each state, we identified a substantial conformational change involving its conserved switch I (sw1) element, which contacts the GTP substrate. By attaching FeBABE (a hydroxyl radical generating probe) to sw1, we could monitor sw1 movement (by approximately 20 A), relative to the 70S ribosome, during the EF-G cycle. In free EF-G, sw1 is disordered, particularly in GDP-bound and nucleotide-free states. On EF-G*GTP binding to the ribosome, sw1 becomes structured and tucked inside the ribosome, thereby locking GTP onto EF-G. After hydrolysis and translocation, sw1 flips out from the ribosome, greatly accelerating release of GDP and EF-G from the ribosome. Collectively, our results support a central role of sw1 in driving the EF-G cycle during protein synthesis.
authors.affiliation: Department of Biochemistry, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
authors.forename: Cristina
authors.initials: C
authors.lastname: Ticu
authors.forename: Roxana
authors.initials: R
authors.lastname: Nechifor
authors.forename: Boray
authors.initials: B
authors.lastname: Nguyen
authors.forename: Melanie
authors.initials: M
authors.lastname: Desrosiers
authors.forename: Kevin S
authors.initials: KS
authors.lastname: Wilson
mesh_terms: D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D008958:Models, Molecular; D020653:Peptide Elongation Factor G / Q000737:chemistry* / Q000378:metabolism*; D010444:Peptide Elongation Factor Tu / Q000378:metabolism; D011487:Protein Conformation; D054680:Ribosome Subunits, Small, Bacterial / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D020653:Peptide Elongation Factor G; D006160:Guanosine Triphosphate; D014357:Trypsin; D010444:Peptide Elongation Factor Tu