Timing of GTP binding and hydrolysis by translation termination factor RF3.
Protein synthesis in bacteria is terminated by release factors 1 or 2 (RF1/2), which, on recognition of a stop codon in the decoding site on the ribosome, promote the hydrolytic release of the polypeptide from the transfer RNA (tRNA). Subsequently, the dissociation of RF1/2 is accelerated by RF3, a guanosine triphosphatase (GTPase) that hydrolyzes GTP during the process. Here we show that--in contrast to a previous report--RF3 binds GTP and guanosine diphosphate (GDP) with comparable affinities. Furthermore, we find that RF3-GTP binds to the ribosome and hydrolyzes GTP independent of whether the P site contains peptidyl-tRNA (pre-termination state) or deacylated tRNA (post-termination state). RF3-GDP in either pre- or post-termination complexes readily exchanges GDP for GTP, and the exchange is accelerated when RF2 is present on the ribosome. Peptide release results in the stabilization of the RF3-GTP-ribosome complex, presumably due to the formation of the hybrid/rotated state of the ribosome, thereby promoting the dissociation of RF1/2. GTP hydrolysis by RF3 is virtually independent of the functional state of the ribosome and the presence of RF2, suggesting that RF3 acts as an unregulated ribosome-activated switch governed by its internal GTPase clock.
Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Frank
F
Peske
Stephan
S
Kuhlenkoetter
Marina V
MV
Rodnina
Wolfgang
W
Wintermeyer
D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D010443:Peptide Chain Termination, Translational; D010454:Peptide Termination Factors / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism
D010454:Peptide Termination Factors; C087971:peptide-chain-release factor 3; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate