Published online 15 September 2005
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Deacylated tRNA is released from the E site upon A site occupation but before GTP is hydrolyzed by EF-Tu
1Max-Planck-Institut für Molekulare Genetik AG Ribosomen, Ihnestrasse 73, D-14195 Berlin, Germany 2Laboratory of Biochemistry, School of Medicine, University of Patras 26500 Patras, Greece
*To whom correspondence should be addressed. Tel: +49 30 8413 1700; Fax: +49 30 8413 1794; Email: nierhaus{at}molgen.mpg.de
Received July 27, 2005. Revised August 29, 2005. Accepted August 29, 2005.
The presence or absence of deacylated tRNA at the E site sharply influences the activation energy required for binding of a ternary complex to the ribosomal A site indicating the different conformations that the E-tRNA imparts on the ribosome. Here we address two questions: (i) whether or not peptidyltransferase—the essential catalytic activity of the large ribosomal subunit—also depends on the occupancy state of the E site and (ii) at what stage the E-tRNA is released during an elongation cycle. Kinetics of the puromycin reaction on various functional states of the ribosome indicate that the A-site substrate of the peptidyltransferase center, puromycin, requires the same activation energy for peptide-bond formation under all conditions tested. We further demonstrate that deacylated tRNA is released from the E site by binding a ternary complex aminoacyl-tRNA•EF-Tu•GDPNP to the A site. This observation indicates that the E-tRNA is released after the decoding step but before both GTP hydrolysis by EF-Tu and accommodation of the A-tRNA. Collectively these results reveal that the reciprocal linkage between the E and A sites affects the decoding center on the 30S subunit, but does not influence the rate of peptide-bond formation at the active center of the 50S subunit.
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