tRNA-ribosome interactions.
Direct measurements of the rates of dissociation of dipeptidyl-tRNA from the ribosome show that hyperaccurate SmP and SmD ribosomes have unstable A-site binding of peptidyl-tRNA, while P-site binding is extremely stable in relation to the wild type. Error-prone Ram ribosomes, on the other hand, have stable A-site and unstable P-site binding of peptidyl-tRNA. At least for these mutant ribosomes, we conclude that stabilization of peptidyl-tRNA in one site destabilizes binding in the other. Elongation factor Tu (EF-Tu) undergoes a dramatic structural transition from its GDP-bound form to its active GTP-bound form, in which it binds aa-tRNA (aminoacyl-tRNA) in ternary complex. The effects of substitution mutations at three sites in domain I of EF-Tu, Gln124, Leu120, and Tyr160, all of which point into the domain I-domain III interface in both the GTP and GDP conformations of EF-Tu, were examined. Mutations at each position cause large reductions in aa-tRNA binding. An attractive possibility is that the mutations alter the domain I-domain III interface such that the switching of EF-Tu between different conformations is altered, decreasing the probability of aa-tRNA binding. We have previously found that two GTPs are hydrolyzed per peptide bond on EF-Tu, the implication being that two molecules of EF-Tu may interact on the ribosome to catalyze the binding of a single aa-tRNA to the A-site. More recently we found that ribosomes programmed with mRNA constructs other than poly(U), including the sequence AUGUUUACG, invariably use two GTPs per peptide bond in EF-Tu function. Other experiments measuring the protection of aa-tRNA from deacylation or from RNAse A attack show that protection requires two molecules of EF-Tu, suggesting an extended ternary complex. To remove remaining ambiguities in the interpretion of these experiments, we are making direct molecular weight determinations with neutron scattering and sedimentation-diffusion techniques.
Ehrenberg
M
M
Department of Molecular Biology, BMC, Uppsala, Sweden.
Bilgin
N
N
Dincbas
V
V
Karimi
R
R
Hughes
D
D
Abdulkarim
F
F
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D008969:Molecular Sequence Data; D010444:Peptide Elongation Factor Tu / Q000378:metabolism*; D012329:RNA, Bacterial / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism*; D015203:Reproducibility of Results; D012270:Ribosomes / Q000378:metabolism*; D012641:Selection, Genetic
D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012343:RNA, Transfer; D010444:Peptide Elongation Factor Tu