Structures of modified eEF2 80S ribosome complexes reveal the role of GTP hydrolysis in translocation.
On the basis of kinetic data on ribosome protein synthesis, the mechanical energy for translocation of the mRNA-tRNA complex is thought to be provided by GTP hydrolysis of an elongation factor (eEF2 in eukaryotes, EF-G in bacteria). We have obtained cryo-EM reconstructions of eukaryotic ribosomes complexed with ADP-ribosylated eEF2 (ADPR-eEF2), before and after GTP hydrolysis, providing a structural basis for analyzing the GTPase-coupled mechanism of translocation. Using the ADP-ribosyl group as a distinct marker, we observe conformational changes of ADPR-eEF2 that are due strictly to GTP hydrolysis. These movements are likely representative of native eEF2 motions in a physiological context and are sufficient to uncouple the mRNA-tRNA complex from two universally conserved bases in the ribosomal decoding center (A1492 and A1493 in Escherichia coli) during translocation. Interpretation of these data provides a detailed two-step model of translocation that begins with the eEF2/EF-G binding-induced ratcheting motion of the small ribosomal subunit. GTP hydrolysis then uncouples the mRNA-tRNA complex from the decoding center so translocation of the mRNA-tRNA moiety may be completed by a head rotation of the small subunit.
Taylor
Derek J
DJ
Howard Hughes Medical Institute, Health Research Inc.,Wadsworth Center, Albany, NY 12201-0509, USA.
Nilsson
Jakob
J
Merrill
A Rod
AR
Andersen
Gregers Rom
GR
Nissen
Poul
P
Frank
Joachim
J
D000244:Adenosine Diphosphate / Q000378:metabolism; D001203:Ascomycota / Q000378:metabolism; D020285:Cryoelectron Microscopy / Q000379:methods; D006160:Guanosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020652:Peptide Elongation Factor 2 / Q000737:chemistry* / Q000378:metabolism; D020653:Peptide Elongation Factor G / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D034443:RNA Transport; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism
D020652:Peptide Elongation Factor 2; D020653:Peptide Elongation Factor G; D012333:RNA, Messenger; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D012343:RNA, Transfer