title: The conserved GTPase HflX is a ribosome splitting factor that binds to the E-site of the bacterial ribosome.
abstract: Using a combination of biochemical, structural probing and rapid kinetics techniques we reveal for the first time that the universally conserved translational GTPase (trGTPase) HflX binds to the E-site of the 70S ribosome and that its GTPase activity is modulated by peptidyl transferase centre (PTC) and peptide exit tunnel (PET) binding antibiotics, suggesting a previously undescribed mode of action for these antibiotics. Our rapid kinetics studies reveal that HflX functions as a ribosome splitting factor that disassembles the 70S ribosomes into its subunits in a nucleotide dependent manner. Furthermore, our probing and hydrolysis studies show that the ribosome is able to activate trGTPases bound to its E-site. This is, to our knowledge, the first case in which the hydrolytic activity of a translational GTPase is not activated by the GTPase activating centre (GAC) in the ribosomal A-site. Furthermore, we provide evidence that the bound state of the PTC is able to regulate the GTPase activity of E-site bound HflX.
authors.affiliation: Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, T1K 3M4, Canada.
authors.forename: Mackenzie L
authors.initials: ML
authors.lastname: Coatham
authors.affiliation: Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, T1K 3M4, Canada.
authors.forename: Harland E
authors.initials: HE
authors.lastname: Brandon
authors.affiliation: Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, T1K 3M4, Canada.
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Fischer
authors.affiliation: Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, T1K 3M4, Canada.
authors.forename: Tobias
authors.initials: T
authors.lastname: Schümmer
authors.affiliation: Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, T1K 3M4, Canada hj.wieden@uleth.ca.
authors.forename: Hans-Joachim
authors.initials: HJ
authors.lastname: Wieden
mesh_terms: D001665:Binding Sites / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000235:genetics* / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D010458:Peptidyl Transferases / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D012270:Ribosomes / Q000201:enzymology / Q000235:genetics*
chemical_list: D029968:Escherichia coli Proteins; D020690:GTPase-Activating Proteins; D010458:Peptidyl Transferases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; C535071:HflX protein, E coli