EF-Tu dynamics during pre-translocation complex formation: EF-Tu·GDP exits the ribosome via two different pathways.
The G-protein EF-Tu, which undergoes a major conformational change when EF-Tu·GTP is converted to EF-Tu·GDP, forms part of an aminoacyl(aa)-tRNA·EF-Tu·GTP ternary complex (TC) that accelerates the binding of aa-tRNA to the ribosome during peptide elongation. Such binding, placing a portion of EF-Tu in contact with the GTPase Associated Center (GAC), is followed by GTP hydrolysis and Pi release, and results in formation of a pretranslocation (PRE) complex. Although tRNA movement through the ribosome during PRE complex formation has been extensively studied, comparatively little is known about the dynamics of EF-Tu interaction with either the ribosome or aa-tRNA. Here we examine these dynamics, utilizing ensemble and single molecule assays employing fluorescent labeled derivatives of EF-Tu, tRNA, and the ribosome to measure changes in either FRET efficiency or fluorescence intensity during PRE complex formation. Our results indicate that ribosome-bound EF-Tu separates from the GAC prior to its full separation from aa-tRNA, and suggest that EF-Tu·GDP dissociates from the ribosome by two different pathways. These pathways correspond to either reversible EF-Tu·GDP dissociation from the ribosome prior to the major conformational change in EF-Tu that follows GTP hydrolysis, or irreversible dissociation after or concomitant with this conformational change.
Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA.
Wei
W
Liu
Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Chunlai
C
Chen
Department of Molecular Biology and Genetics and Interdisciplinary Nanoscience Center (iNANO), Aarhus University, DK-8000 Aarhus C, Denmark.
Darius
D
Kavaliauskas
Department of Molecular Biology and Genetics and Interdisciplinary Nanoscience Center (iNANO), Aarhus University, DK-8000 Aarhus C, Denmark.
Charlotte R
CR
Knudsen
Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Yale E
YE
Goldman
Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA cooprman@pobox.upenn.edu.
Barry S
BS
Cooperman
D031541:Fluorescence Resonance Energy Transfer; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D010441:Peptide Chain Elongation, Translational; D010444:Peptide Elongation Factor Tu / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*
D012346:RNA, Transfer, Amino Acyl; D012269:Ribosomal Proteins; C027624:ribosomal protein L11; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu