title: Identification of two structural elements important for ribosome-dependent GTPase activity of elongation factor 4 (EF4/LepA).
abstract: The bacterial translational GTPase EF4/LepA is structurally similar to the canonical elongation factor EF-G. While sharing core structural features with other translational GTPases, the function of EF4 remains unknown. Recent structural data locates the unique C-terminal domain (CTD) of EF4 in proximity to the ribosomal peptidyl transferase center (PTC). To investigate the functional role of EF4's CTD we have constructed three C-terminal truncation variants. These variants are fully functional with respect to binding mant-GTP and mant-GDP as determined by rapid kinetics, as well as their intrinsic multiple turnover GTPase activity. Furthermore, they are able to form stable complexes with the 70S ribosome and 50S/30S ribosomal subunits. However, successive removal of the C-terminus impairs ribosome-dependent multiple turnover GTPase activity of EF4, which for the full-length protein is very similar to EF-G. Our findings suggest that the last 44 C-terminal amino acids of EF4 form a sub-domain within the C-terminal domain that is important for GTP-dependent function on the ribosome. Additionally, we show that efficient nucleotide hydrolysis by EF4 on the ribosome depends on a conserved histidine (His 81), similar to EF-G and EF-Tu.
authors.affiliation: Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, AB, T1K 3M4, Canada.
authors.forename: Evelina Ines
authors.initials: EI
authors.lastname: De Laurentiis
authors.affiliation: Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, AB, T1K 3M4, Canada.
authors.forename: Hans-Joachim
authors.initials: HJ
authors.lastname: Wieden
mesh_terms: D055162:Biocatalysis; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D016296:Mutagenesis; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; C515651:LepA protein, E coli; D010448:Peptide Initiation Factors; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate