Abstract
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.
Highlights
EF-Tu is, along with EF-G, one of two G-protein factors that are required for nascent polypeptide elongation by the prokaryotic 70S ribosome [1]
Rapid mixing of TCQSY9 with a 70SICCy3 programmed with mRNA022 [19], which has a UUU codon following the AUG initiation codon, leads to a biphasic change in Cy3 fluorescence, with an initial decrease corresponding to ternary complex (TC) binding to the ribosome that places EF-Tu position 348 in proximity to L11 position 87, followed by a restoration of fluorescence intensity as these two positions move apart [15]
It follows from this observation that the 3 -end of aa-tRNA must migrate from its binding site within EF-Tu while it is in the A/T site [ndb 4V5L [29]], to the A-site on the 50S subunit [ndb 4V5D [35]], a distance of some 80 A, prior to EF-Tu dissociation from the ribosome
Summary
EF-Tu is, along with EF-G, one of two G-protein factors that are required for nascent polypeptide elongation by the prokaryotic 70S ribosome [1]. In the first elongation cycle, EF-Tu binds as part of an aminoacyl(aa)-tRNA·EFTu·GTP ternary complex (TC) to the 70S initiation complex (70SIC), which contains initiator fMet-tRNAfMet bound in the P-site. Formation of base pairs between the anticodon loop of aatRNA and the cognate codon triplet in mRNA activates EF-Tu GTPase activity. This is followed by GTP hydrolysis, Pi release, aa-tRNA accommodation into the A-site, peptide bond formation, and, EF-Tu·GDP dissociation from the ribosome, resulting in pretranslocation (PRE) complex formation. EF-G·GTP catalysis of the translocation of fMet-aa-tRNA and tRNAfMet from the A- and Psites to the P- and E-sites, respectively, along with their bound mRNA codons, completes the first elongation cycle by the formation of the posttranslocation (POST) complex
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