Deacylated tRNA is released from the E site upon A site occupation but before GTP is hydrolyzed by EF-Tu.
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.
Dinos
George
G
Max-Planck-Institut für Molekulare Genetik AG Ribosomen, Ihnestrasse 73, D-14195 Berlin, Germany.
Kalpaxis
Dimitrios L
DL
Wilson
Daniel N
DN
Nierhaus
Knud H
KH
D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008957:Models, Genetic; D010441:Peptide Chain Elongation, Translational; D010444:Peptide Elongation Factor Tu / Q000378:metabolism*; D010458:Peptidyl Transferases / Q000378:metabolism*; D011691:Puromycin / Q000494:pharmacology; D012343:RNA, Transfer / Q000378:metabolism*; D012270:Ribosomes / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D046249:Transfer RNA Aminoacylation
D011691:Puromycin; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D010458:Peptidyl Transferases; D010444:Peptide Elongation Factor Tu