ABSTRACT
Equilibrium binding affinity of yeast tRNA(Phe) for Escherichia coli poly(U)-programmed 70S ribosomal P-site was compared with corresponding affinities of several tRNA(Phe) 3'- and 5'-end-truncated derivatives, all containing the anticodon arm. Our findings strongly suggest that besides three 3'-terminal-CCA nucleotides (C74, C75 and A76), only the tRNA(Phe) anticodon arm (N28-N42) contains ribosomal P-site contact centers and that there are no such centers in the intermediate regions N1-N27 and N43-N73.
Subject(s)
Escherichia coli/metabolism , Poly U/metabolism , RNA, Ribosomal/metabolism , RNA, Transfer, Phe/metabolism , Ribosomes/metabolism , Saccharomyces cerevisiae/metabolism , Anticodon , Base Sequence , Binding Sites , Escherichia coli/genetics , Kinetics , Molecular Sequence Data , Oligoribonucleotides/chemistry , Saccharomyces cerevisiae/geneticsABSTRACT
Poli(U)-dependent binding of yeast tRNA(Phe) lacking the 3'-terminal CA or CCA residues, and fragments of tRNA prepared by splitting the tRNA(Phe) at the G18 or 7mG46 nucleotides, to the 70S ribosomal P-site was studied. Equilibrium binding constants and free energies of binding were measured. The contribution of C74 and C75 was estimated at 3.9 kJ/mol and 3.0 kJ/mol, respectively, at 10 mM Mg2+ and 30 degrees C. The free energies of the binding of the anticodon arm the tRNA lacking its CCA terminus, and the tRNA fragment containing bases N1-N45 were found to be nearly equal. These results indicate that only the anticodon arm and the CCA terminus take part in deacylated tRNA-P-site interaction.
Subject(s)
Escherichia coli/metabolism , Poly U/metabolism , RNA, Transfer, Phe/metabolism , Ribosomes/metabolism , Saccharomyces cerevisiae/metabolism , Anticodon , Binding Sites , ThermodynamicsABSTRACT
The poly(U)-dependent bindings of yeast tRNAPhe, its derivative depleted of 3'-terminal adenosine, and 15-nucleotide having a sequence of yeast tRNAPhe anticodon arm to the P site of Escherichia coli 70S ribosomes were compared. The equilibrium and rate constants were determined. Data indicate that the anticodon arm (N28-N42) contributes the major fraction of the binding free energy (-45.3 kJ/mol at 10 mM Mg2+ and 30 degrees C). Other parts of the tRNAPhe molecule besides A76 (N1-N27 and N43-N75) bring additional-6.0 kJ/mol, and A76 contributes-2.4 kJ/mol.
Subject(s)
Escherichia coli/cytology , Poly U/metabolism , RNA, Transfer, Phe/metabolism , Ribosomes/metabolism , Yeasts/metabolism , Kinetics , ThermodynamicsABSTRACT
In the presence of methanol 50S ribosomal subunits reveal two independents sites for binding of deacylated tRNA and/or AcPhe-tRNA. The site with lower affinity was identified with the donor (P') site as the dissociation constant (Ka) for AcPhe-tRNA was equal to the Michaelis constant for its reaction with puromycin both at 0 degrees C and 25 degrees C. Log Ka increases linearly with methanol concentration. This suggests that there are no conformational transitions of the interacting components, the affinity increases only quantatively due to lowering of the dielectric constant of water, and the site can exist even in the absence of methanol, but its Ka may be too low to be measured. It follows from these data that the higher-affinity site, which is observed both in the absence and presence of methanol, cannot be the P' site as it was generally believed. By all its properties it is more like the additional E site, which has been recently found on 70S ribosomes. Specifically, its affinity for deacylated tRNA is about 1000-fold higher than for AcPhe-tRNA (in the P'-site they are almost the same).
