title: Labeled EF-Tus for rapid kinetic studies of pretranslocation complex formation.
abstract: The universally conserved translation elongation factor EF-Tu delivers aminoacyl(aa)-tRNA in the form of an aa-tRNA·EF-Tu·GTP ternary complex (TC) to the ribosome where it binds to the cognate mRNA codon within the ribosomal A-site, leading to formation of a pretranslocation (PRE) complex. Here we describe preparation of QSY9 and Cy5 derivatives of the variant E348C-EF-Tu that are functional in translation elongation. Together with fluorophore derivatives of aa-tRNA and of ribosomal protein L11, located within the GTPase associated center (GAC), these labeled EF-Tus allow development of two new FRET assays that permit the dynamics of distance changes between EF-Tu and both L11 (Tu-L11 assay) and aa-tRNA (Tu-tRNA assay) to be determined during the decoding process. We use these assays to examine: (i) the relative rates of EF-Tu movement away from the GAC and from aa-tRNA during decoding, (ii) the effects of the misreading-inducing antibiotics streptomycin and paromomycin on tRNA selection at the A-site, and (iii) how strengthening the binding of aa-tRNA to EF-Tu affects the rate of EF-Tu movement away from L11 on the ribosome. These FRET assays have the potential to be adapted for high throughput screening of ribosomal antibiotics.
authors.affiliation: Department of Chemistry and ‡Pennsylvania Muscle Institute, School of Medicine, University of Pennsylvania , Philadelphia, Pennsylvania 19104, United States.
authors.forename: Wei
authors.initials: W
authors.lastname: Liu
authors.forename: Darius
authors.initials: D
authors.lastname: Kavaliauskas
authors.forename: Jared M
authors.initials: JM
authors.lastname: Schrader
authors.forename: Kiran
authors.initials: K
authors.lastname: Poruri
authors.forename: Victoria
authors.initials: V
authors.lastname: Birkedal
authors.forename: Emanuel
authors.initials: E
authors.lastname: Goldman
authors.forename: Hieronim
authors.initials: H
authors.lastname: Jakubowski
authors.forename: Wlodek
authors.initials: W
authors.lastname: Mandecki
authors.forename: Olke C
authors.initials: OC
authors.lastname: Uhlenbeck
authors.forename: Charlotte R
authors.initials: CR
authors.lastname: Knudsen
authors.forename: Yale E
authors.initials: YE
authors.lastname: Goldman
authors.forename: Barry S
authors.initials: BS
authors.lastname: Cooperman
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000502:physiology*; D011487:Protein Conformation; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry* / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000502:physiology*
chemical_list: D029968:Escherichia coli Proteins; D012346:RNA, Transfer, Amino Acyl; D012269:Ribosomal Proteins; C027624:ribosomal protein L11; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu