Single-molecule structural dynamics of EF-G--ribosome interaction during translocation.
EF-G catalyzes translocation of mRNA and tRNAs within the ribosome during protein synthesis. Detection of structural states in the reaction sequence that are not highly populated can be facilitated by studying the process one molecule at a time. Here we present single-molecule studies of translocation showing that, for ribosomes engaged in poly(Phe) synthesis, fluorescence resonance energy transfer (FRET) between the G' domain of EF-G and the N-terminal domain of ribosomal protein L11 occurs within two rapidly interconverting states, having FRET efficiencies of 0.3 and 0.6. The antibiotic fusidic acid increases the population of the 0.6 state, indicating that it traps the ribosome.EF-G complex in a preexisting conformation formed during translation. Only the 0.3 state is observed when poly(Phe) synthesis is prevented by omission of EF-Tu, or in studies on vacant ribosomes. These results suggest that the 0.6 state results from the conformational lability of unlocked ribosomes formed during translocation. An idling state, possibly pertinent to regulation of protein synthesis, is detected in some ribosomes in the poly(Phe) system.
Wang
Yuhong
Y
Pennsylvania Muscle Institute, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6083, USA.
Qin
Haiou
H
Kudaravalli
Rama D
RD
Kirillov
Stanislas V
SV
Dempsey
Graham T
GT
Pan
Dongli
D
Cooperman
Barry S
BS
Goldman
Yale E
YE
D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes / Q000378:metabolism; D005672:Fusidic Acid / Q000737:chemistry / Q000494:pharmacology*; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D020653:Peptide Elongation Factor G / Q000737:chemistry / Q000378:metabolism*; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000378:metabolism; D014176:Protein Biosynthesis; D011487:Protein Conformation; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000737:chemistry / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000737:chemistry / Q000378:metabolism*; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics
D005456:Fluorescent Dyes; D020653:Peptide Elongation Factor G; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012360:RNA, Transfer, Phe; D012269:Ribosomal Proteins; C027624:ribosomal protein L11; D006153:Guanosine Diphosphate; D005672:Fusidic Acid; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D010444:Peptide Elongation Factor Tu